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Protein Folding - Anfinsen's Experiment
- Protein folding refers to the set of ordered pathways by which protein folds into their native functional confirmation.
- Protein folding is primarily driven by hydrophobic forces.
| Anfinsen's Experiment |
- The first insight to this question was provided by Christian Anfinsen at the NIH. He was working on the properties of ribonuclease A (a single chain protein of 124 amino acids with 4 di-sulphide bonds). He unfolded (denatured) ribonuclease A using urea and mercaptoethanol (denaturants). The protein lost its function. Then he allowed to renature ribonuclease A by removing denaturants, and found out that ribonuclease A folded spontaneously and become functional. He concluded that Ribonuclease A can self assemble into its 3D functional structure.
- Primary structure dictates the 3D structure of protein Or primary structure has the program or code for forming a properly folded functional protein.
- Inside the cell, protein folding is assisted by different proteins collectively called as accessory proteins.
- Protein folding assisting proteins or accessory proteins .
- to engineer novel protein
- to get better insight into diseases associated with protein folding
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Protein folding in the cell: an inside story
Nature Medicine volume 17 , pages 1211–1216 ( 2011 ) Cite this article 3698 Accesses 30 Citations 4 Altmetric Metrics details
The final step of information transfer from DNA to effector protein involves the folding of newly translated polypeptide chains into characteristic three-dimensional active structures. In the fall of 1972, while working in a biochemistry laboratory as an undergraduate at Brown University, I heard about an astonishing experiment for which Christian Anfinsen was receiving the Nobel Prize in Chemistry. Anfinsen and his co-workers had unfolded purified RNase A by reducing its disulfide bonds and exposing it to a denaturing agent, and then asked whether the protein could find its way back to the enzymatically active native state upon removal of the reducing agent and denaturant 1 . Amazingly, it did. I was utterly haunted by the beauty of this observation and by the profound conclusion that the primary structure of a protein contains all the information necessary to direct its folding to the native functional state. I could never have imagined that, many years later and in a completely unexpected fashion, I would have something to add to something so fundamentally beautiful. I finished my undergraduate and medical training at Brown and went to Yale for pediatric residency, but, by the second year of training, I was missing the laboratory. I became captivated by the problem of malignant transformation mediated by single viral genes, and, by the end of my residency, I had amassed on my nightstand a stack of several hundred papers on the topic. This is a preview of subscription content, access via your institution Access optionsSubscribe to this journal Receive 12 print issues and online access 195,33 € per year only 16,28 € per issue Buy this article
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Download references AcknowledgementsMany of the major participants in the chaperonin work referred to above are pictured in the illustration on p. xiii. There have been many other collaborators, both in the early work and more recently, who also contributed substantially to the understanding of this system. I regret that space limitations prevented me from referring to them here, but I want to express how deeply grateful I am to everyone with whom I've interacted. Surely, the recognition of this work is shared by all of us. But, more selfishly, it has been a pure joy for me over these past 20 years to work in the lab, at the bench, day by day and side by side with my group members, sharing our ideas, dreams, reagents, frustrations and, of course, joys of discovery as a scientific family. No one could ask for a more enjoyable life. I wish to thank the US National Institutes of Health for supporting the early phase of our work and the Howard Hughes Medical Institute (HHMI) for supporting our subsequent work. I am particularly grateful to the HHMI for allowing me to 'follow my nose' through this work, no matter how risky the undertaking. I also thank HHMI for making our work environment a paradise in which to pursue ideas and do experiments. Finally, I thank W. Fenton for his critical comments during the preparation of this manuscript. Author informationAuthors and affiliations. Arthur L. Horwich is at the Yale University School of Medicine, New Haven, Connecticut, USA., Arthur L Horwich You can also search for this author in PubMed Google Scholar Corresponding authorCorrespondence to Arthur L Horwich . Ethics declarationsCompeting interests. The author declares no competing financial interests. Rights and permissionsReprints and permissions About this articleCite this article. Horwich, A. Protein folding in the cell: an inside story. Nat Med 17 , 1211–1216 (2011). https://doi.org/10.1038/nm.2468 Download citation Published : 11 October 2011 Issue Date : October 2011 DOI : https://doi.org/10.1038/nm.2468 Share this articleAnyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative This article is cited byUnfolding a molecular trefoil derived from a zwitterionic metallopeptide to form self-assembled nanostructures. Nature Communications (2015) Identification of microRNAs and their targets in tomato infected with Cucumber mosaic virus based on deep sequencing
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Part of the book series: SpringerBriefs in Molecular Science ((BRIEFSPROTEIN)) 1867 Accesses 7 Citations We have come a long way since coining of the term protein and the early findings that proteins are charged macromolecules composed of strings of amino acids linked by peptide bonds. Today, structural biologists have technologies that allow in many cases to achieve an atomic-level understanding of protein structure, dynamics and folding; protein physics approaches have made substantial contributions to understanding the intricacies of folding mechanisms and its energetics; biochemists have developed conceptual frameworks to relate protein structure with biological functions. Yet, despite the efforts of a vibrant community of protein scientists, a lot of questions remain to be answered in the field of protein structure and folding. This is a preview of subscription content, log in via an institution to check access. Access this chapter
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Rights and permissionsReprints and permissions Copyright information© 2019 The Author(s), under exclusive license to Springer Nature Switzerland AG About this chapterGomes, C.M., Faísca, P.F.N. (2019). Protein Folding: An Introduction. In: Protein Folding. SpringerBriefs in Molecular Science(). Springer, Cham. https://doi.org/10.1007/978-3-319-00882-0_1 Download citationDOI : https://doi.org/10.1007/978-3-319-00882-0_1 Published : 26 February 2019 Publisher Name : Springer, Cham Print ISBN : 978-3-319-00881-3 Online ISBN : 978-3-319-00882-0 eBook Packages : Biomedical and Life Sciences Biomedical and Life Sciences (R0) Share this chapterAnyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative
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How to Print an Organ from ScratchRenewable energy as an opportunity for all, openmind books, scientific anniversaries, the higgs boson and the origin of mass, featured author, latest book, anfinsen and the architecture of proteins. If each of the proteins that make up living beings were an origami figure, the instructions for folding them in three dimensions would have to be sought in their amino-acid sequence. The first test of this scientific principle, which is now found in all biology textbooks, was provided in 1961 by an American biochemist named Christian B. Anfinsen (1916-1995), a descendant of Norwegian immigrants, who worked at the United States National Institutes of Health and years later would be awarded a Nobel Prize. Anfinsen wanted to understand what the spatial organization, or 3D shape, of a protein depended on, essential for its correct functioning. Searching in his experiments for a single molecule whose activity was easily measurable, he opted for ribonuclease, an enzyme that breaks RNA into fragments. And to study it, he had an idea that no one had thought of before: unfold the protein and then test different ways to refold it. Unfolding a protein involves breaking the molecular bonds, thereby causing the loss of the natural structure of the protein (denaturation) and its biological properties. Anfinsen accomplished this by mixing mercaptoethanol and urea, and the protein changed from being a complex three-dimensional figure to a long stretched chain. The question was whether it would be a reversible change, and if so, would it refold itself exactly the same?  The American biochemist soon answered the question. When the optimum environmental conditions were restored, then voila! , the protein returned to its original shape and recovered 100% of its activity. It was impossible that it was by random chance that it always formed the exact same four original links when refolding itself. Without doubt, some unique instructions existed on how to fold it, and they had to be contained within the protein’s own sequence. The primary structure, he concluded, is all that a protein needs for its correct folding. He had guessed correctly, this mature scientist who was mistaken as a teenager when he believed that all his schoolmates were geniuses except him. Anfinsen had much to contribute to molecular biology and genetics. In fact, he did not stop there in his research on ribonuclease. Fulfilling Einstein’s maxim that “the important thing is not to stop questioning,” he decided to find out why it was that, among the 104 possible options for folding this enzyme, nature opts for one and not any other. He concluded that it was, essentially, a matter of energy savings. In other words, among all possible configurations, all proteins will opt for the one that consumes the least energy within the cell. This is known as Anfinsen’s dogma, which earned him the Nobel Prize in Chemistry in 1972. It later came to be understood that there exist exceptions to this dogma, called prions, which cause the so-called “mad cow disease” (bovine spongiform encephalopathy), which become infectious precisely because of changes in their three dimensional structure.  For the rest of his career, this biochemist and music aficionado —he played the viola and piano for relaxation—mainly devoted himself to developing chromatographic sequencing techniques that permitted the reading of the “code” of many proteins, as well as understanding the workings of some unique living beings, hyperthermophilic bacteria, which live at extremely high temperatures, near the boiling point, a seemingly unimaginable place for the presence of life. After living through the horrors of World War II during a stay in Denmark, he became an advocate for nuclear disarmament and human rights. During the Cold War, he defended the scientific collaboration between the US and the Soviet Union. Anfinsen was one of the Nobel laureates who expressed his opposition in 1973 to US President Richard Nixon’s decision to give priority to research on cancer. Later, he publicly opposed the science policy of Reagan, who applied harsh cuts to biomedical research. And he was also involved in a rescue mission to Argentina in 1981, to release twelve scientists detained by the military government of dictator Jorge Rafael Videla. In addition to his political activism, he contributed interesting reflections on the relationship between science and religion. In an exchange of correspondence with physicist Henry Margenau, Anfinsen clarified that although for him the origin of the universe was explained by the Big Bang, and the origin of life was “an inevitable consequence of the evolution of the universe,” he did not consider this incompatible with having religious beliefs. “Religion seeks mystical answers and science responds to human curiosity by means of physical laws that govern the world,” he argued. Regarding his thoughts about the existence of God, he said, “I think only an idiot can be an atheist. We must admit that there is a power or incomprehensible force with unlimited knowledge that launched the universe in the first place. […] It seems obvious that no one will ever understand what actually happens and that the best we can do is to relax and enjoy ourselves.” Elena Sanz for Ventana al ConocimientoRelated publications.
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Learning Resource TypesProtein folding and human disease, anfinsen experiments. This resource contains information regarding study materials.  You are leaving MIT OpenCourseWare Scheme of Anfinsen's experiment. See text for details Similar publications
Oxford Protein Informatics GroupOr "opig" to friends.  Expanding Anfinsen’s Principle (Journal Club)Paper : Expanding Anfinsen’s Principle: Contributions of Synonymous Codon Selection to Rational Protein Design. In 1961, Anfinsen performed his now (in)famous denaturing experiment upon ribonuclease A, a small one hundred residue globular protein. He showed that it could both unfold and refold via the addition and subsequent removal of chemical substances. From this he concluded that a protein’s fold is that of its global free energy minimum and, consequently, all the information required to know the folded structure of a given protein is solely encoded within its sequence of amino acids. In 1972, Anfinsen was awarded the Nobel prize for this work from which stemmed the vast field of protein folding prediction, a global arms race to see who could best predict/find the elusive global minimum for any given protein. Unfortunately, protein fold prediction is still in its infancy with regards to its predictive power. As a scientific community, we have made huge progress using homology models, whereby we use the structure of a protein with similar sequence to the one under investigation to provide a reasonable starting point for refinement. However, when there is no similar structure in existence, we struggle abysmally due to being forced to resort to de novo models. This lack of ability when we are given solely a sequence to work with, shows that that our fundamental understanding of the protein folding process must be incomplete. An increasingly common viewpoint, one that is at odds with Anfinsen’s conclusions, is that there is additional information required for a protein to fold. One suggested source of information is in the production of the protein itself at the ribosome. Coined as cotranslational folding, it has been shown that a protein undergoing synthesis will fold as it emerges from the ribosome, not waiting until the entire sequence is synthesised. As such, the energy landscape that the protein must explore to fold is under constant change and development as more and more of the protein emerges from the ribosome. It is an iterative process of smaller searches as the energy landscape is modulated in steps to that of the complete amino acid sequence. Another suggested source of information is within the degeneracy observed within the genetic code. Each amino acid is encoded for by up to 6 different codons, and as such, one can never determine exactly the coding DNA that created a given protein. While this degeneracy has been suggested as merely an effect to reduce the deleterious nature of point mutations, it has also been found that each of these codons are translated at a different rate. It is evident that information is consumed when RNA is converted into protein at the ribosome, sine reverse translation is impossible, and it is hypothesised that these variations in speed can alter the final protein structure.  Figure 1. Experimental design for kinetically controlled folding. (a) Schematic of YKB, which consists of three half-domains connected by flexible (AGQ)5 linkers (black lines). The Y (yellow) and B (blue) half-domains compete to form a mutually exclusive kinetically trapped folded domain with the central K (black) half-domain. The red wedge indicates the location of synonymous codon substitutions (see text). (b) Energy landscapes for proteins that fold under kinetic control have multiple deep minima, representing alternative folded structures, separated by large barriers. The conformations of the unfolded protein and early folding intermediates (colored arrows) determine the final folded state of the protein. Forces that constrict the unfolded ensemble (grey cone) can bias folding toward one structure. (c) During translation of the nascent chain by the ribosome (orange), folding cannot be initiated from the untranslated C-terminus, which restricts the ensemble of unfolded states and leads to the preferential formation of one folded structure. Image sourced from J. Am. Chem. Soc., 2014, 136(3), The journal club paper by Sander et al. looked experimentally at whether both cotranslational folding and codon choice can have effect on the resultant protein structure. This was achieved through the construction of a toy model protein, consisting of three half domains as shown in Figure 1. Each of these half domains were sourced from bifluorescent proteins, a group of protein half domains that when combined fluoresce. The second half domain (K) could combine with either the first (Y) or the third (B) half domains to create a fluorophore, crucially this occurring in a non-reversible fashion such that once a full domain was formed it could not form the other. By choosing flurophores that differed in wavelength, it was simple to measure the ratio in which the species, YK-B or Y-KB, were formed. They found that the ratio of these two species differed between in-vitro and in-vivo formation. When denatured Y-K-B species were allowed to refold, a racemic mixtrue was produced, both species found the be equally likely to form. In contrast, when synthesised at the ribosome, the protein showed an extreme bias to form the YK-B species as shown in Figure 2. They concluded that this is caused by cotranslational folding, the half domains Y and K having time to form the YK species before B was finished being produced. As pointed out by some members within the OPIG group, it would have been appreciated to see if similar results were produced if the species were reversed, such that B was synthesised first and Y last, but this point does not invalidate what was reported.  Figure 2. Translation alters YKB folded structure. (a) Fluorescence emission spectra of intact E. coli expressing the control fluorescent protein constructs YK (yellow) or KB (cyan). (b) Fluorescence emission spectra of intact E. coli expressing YKB constructs with common or rare codon usage (green versus red solid lines) versus the same YKB constructs folded in vitro upon dilution from a chemical denaturant (dashed lines). Numbers in parentheses correspond to synonymous codon usage; larger positive numbers correspond to more common codons. (c) E. coli MG1655 relative codon usage(3) for codons encoding three representative YKB synonymous mutants: (+65) (light green), (−54) (red), and (−100) (pink line). Image sourced from J. Am. Chem. Soc., 2014, 136(3). Following the above, they also probed the role of codon choice using this toy model system. They varied the codons choice over a small segment of residues between the K and B half domains, such that the had multitude of species which would be encoded either “faster” or “slower” across this region. Codon usage was used as the measure of speed, though this has yet to established within the literature as to its appropriateness. They found that the slower species increased the bias towards the YK-B over Y-KB, while faster species reduced it. This experiment shows clearly that codon choice has a role on a protein’s final structure, though they only show a large global effect. My work is primarily on whether codon choice has a role at the secondary structure level, so I will be avidly hoping that more experiments will follow that show the role of codons at finer levels. In conclusion, Sander et al. performed one of the cleanest experimental proofs of cotranslational folding to date. Older evidence is more anecdotal in nature, with reports of protein X or Y changing in response to a single synonymous mutation. In contrast, the experiment reported here is systematic in the approach and leaves little room for doubt over the results. Secondly and more ground breaking, is the (again) systematic nature in which codon choice is investigated and shown to effect the global protein structure. This is one of those rare pieces of science which the conclusions are clear and forthcoming to all readers.  Login to your accountIf you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
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Molecular Chaperones: Opening and closing the Anfinsen cage
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 The Anfinsen Dogma: Intriguing Details Sixty-Five Years LaterGiorgia gambardella. 1 Department of Chemical Sciences and Technologies, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, 00133 Rome, Italy; [email protected] (G.G.); [email protected] (S.N.); moc.liamg@oiradarretavac (D.C.); ti.2amorinu@10ssldcb (A.B.) Sara NotariDario cavaterra, federica iavarone. 2 Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; [email protected] 3 Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00168 Rome, Italy Massimo Castagnola4 Laboratorio di Proteomica, Centro Europeo di Ricerca sul Cervello, IRCCS Santa Lucia, 00179 Rome, Italy; ti.kooltuo@alongatsacxam Alessio BocediGiorgio ricci, associated data. Data are contained within the article. The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role of the correct disulfide in the attainment of the native structure. CD analysis, mass spectrometry, fluorescence spectroscopy and enzyme activity indicate that native disulfides drive the formation of the secondary and tertiary structures that cannot be entirely formed in their absence. This opposes a common opinion that these structures are first formed and then stabilized by the native disulfides. Our results also indicate that a spontaneous re-oxidation of a reduced RNase cannot produce a complete recovery of activity, as described by many textbooks; this can be obtained only in the presence of a reshuffling solution such as GSH/GSSG. 1. IntroductionThe pioneering study of Anfinsen about the denaturation and renaturation of ribonuclease A (RNase) represents one of the most important milestones for the understanding of protein folding, and his conclusions reported in all textbooks of biochemistry are known as the “ Anfinsen dogma ” [ 1 , 2 , 3 ]. Briefly, he says that at conditions at which folding occurs, the native structure is a unique, stable and kinetically accessible minimum of the free energy. In other terms, the native structure is uniquely determined by the primary structure [ 4 ]. As reported by almost all textbooks, two historical experiments made it possible to prove this fundamental property of proteins, i.e., a complete inactive RNase with scrambled disulfides recovers its native activity and the correct disulfides upon incubation with catalytic amount of β-mercaptoethanol (β-ME), which allows a productive reshuffling [ 5 ]. A second finding is that a reduced and inactive RNase, obtained in the presence of β-ME and 8 M urea, recovers its full native structure and activity by removing these reagents by dialysis and using atmospheric oxygen as oxidizing agent [ 1 , 2 , 3 ]. Over the past forty years, my colleagues and I have described these two experiments to our students, exactly as reported above and by almost all textbooks of biochemistry, but when we proposed them to read the original articles, their research gave curious results. Surprisingly, we did not find any papers by Anfinsen describing the removal of urea and β-ME using dialysis, but only by means of gel filtration [ 6 , 7 ]. This appeared to us a no slight difference, since gel filtration causes a very fast separation of urea and β-ME from RNase, while dialysis is a very slow process that makes it possible for a residual amount of the reducing compound to reshuffle incorrect disulfides. More importantly, we noted that the first results obtained after spontaneous re-oxidation of reduced RNase indicated that only 20% of its native activity was recovered starting from a full reduced enzyme [ 8 ]. Only a few years later, an almost complete re-activation (80–100%) has been described using particular experimental conditions (i.e., very low protein concentrations, non-physiological temperature, etc.) [ 9 ]. Thus, the complete recovery of the native structure seems to require particular conditions and to be a not so univocal and simple process as described in all biochemistry textbooks [ 1 , 2 , 3 ]. Furthermore, very scarce is the knowledge about the fate of secondary structures during the oxidative folding and during the reductive unfolding of RNase, because one of the most detailed circular dichroism (CD) spectral studies was performed on modified enzyme (carboxymethylated and carboxyamidomethylated) and using not advanced curve-fitting analysis of CD data [ 10 ]. The current availability of more accurate and simplified RNase activity methods, fluorescence and mass spectrometry data together with software for the quantitative estimation of secondary structures from CD spectra, encouraged us to reproduce some RNase folding/unfolding experiments to recover additional information. As it will be clear below, the “ Anfinsen dogma ” [ 4 ] will always remain intact, but sixty-five years later a few intriguing details must be refined and corrected, opening surprising conclusions about the role of disulfides in the oxidative folding of this enzyme. We underline that the current work is not a comprehensive reassessment of the “ Anfinsen dogma ”. Rather, it is a study of how experimental conditions dictate the outcome of the refolding of RNase A in vitro. We are also aware that RNase A could exhibit different separable forms enzyme structures able to perform different activities [ 11 ], but even these aspects have not been considered in our investigation. 2.1. Does the Reduced and Denatured RNase Spontaneously Recover Its Native Structure and Activity?In the pioneering report by Sela and co-workers [ 8 ], these authors reported that totally reduced RNase obtained using thioglycolic acid as reducing agent and 8 M urea as denaturant, after removal of these reagents and oxidation at pH 7.0 or 8.0 by air bubbling at room temperature (25 °C), no more than 12–19% than the original activity can be restored. A more efficient but not complete recovery of activity was obtained (55% of the native activity) starting from a partially reduced enzyme with one intact native disulfide [ 8 ]. In a meticulous study by White [ 12 ], eight samples of RNase were reduced using a purified sample of thioglycolic acid. The re-oxidation process gave 85% as mean of reactivation. However, only 50% of the original soluble enzyme was recovered after lyophilization, indicating that relevant amounts of oxidized RNase acquired scarce solubility probably due to incorrect inter- or intra-molecular disulfides. Thus, taking into account this loss, an amount of native and active enzyme of 43% that was formed through this procedure is reasonable. Very similar results were obtained using β-ME as reducing agent [ 12 ]. Only a few years later, Anfinsen described an almost complete recovery of activity (80–100%) starting from a full reduced RNase with β-ME in 8 M urea, removal this reagent with a Sephadex G-25 column, and subsequent exposition to air (20 h) at low RNase concentrations (~25 µM) at pH 8.0–8.5 [ 9 ]. Our attempts to reproduce these results were disappointing in both kinetics of disulfide formation and activity recovery under various temperature and protein concentrations. More precisely, starting from the reduced and denatured RNase I (rRNase I and other rRNase samples, i.e., rRNase II, III and IV, differ mainly in the chromatographic purification step, see Materials and Methods), incubation at pH 8.5 with very low protein concentration (14 µM) did not give a complete oxidation before two days had elapsed ( Table 1 ), and no more than 20–30% of the original activity ( Figure 1 A and Table 1 ). We believe that the faster oxidation observed by Anfinsen and Haber (20 h) [ 9 ] was possibly due to an amount of spurious metals, a not uncommon flaw in buffer preparations in the mid-20th century and, in traces, even present in our experiments ( Figure 1 A). In fact, EDTA strongly inhibits the re-oxidation and the presence of trace of Cu 2+ and other metals greatly enhances the process as also observed by other authors ( Figure 1 B) [ 10 , 13 ]. The presence of 10 µM Cu 2+ gave very low recovery of activity compared to the 0.3 µM Cu 2+ possibly due to a fast and tumultuous disulfide formation (mostly incorrect).  Re-oxidation of rRNase I (14 µM) in 20 mM Tris-HCl pH 8.5 under different conditions. ( A ) Recovery of activity after incubation at 37 °C without (orange column), or in the presence of Cu 2+ (10 µM) (light blue column). At 25 °C in presence of Cu 2+ (10 µM) (grey column), Cu 2+ (1 µM) (pink column), Cu 2+ (0.3 µM) (green column), and Cu 2+ (0.3 µM) with β-ME (11 µM) (red column). All the activities are compared to the activity of the native protein (black column). Activities were measured as described under Materials and Methods. The error bars represent the S.D. derived from three independent experiments. ( B ) Time-dependent disappearance of rRNase I sulfhydryl groups in 20 mM Tris-HCl pH 8.5, at 25 °C under different conditions: rRNase I (14 µM) with Cu 2+ (0.3 µM) (green line), with Cu 2+ (1 µM) (pink line), with Cu 2+ (10 µM) (grey line), and with only EDTA (0.5 mM) (black line). The error bars represent the S.D. derived from three independent experiments. ( C ) CD spectra of rRNase I at the end of the re-oxidation process. Native RNase (black line), re-oxidized rRNase I at 37 °C (orange line), re-oxidized rRNase I at 37 °C in the presence of Cu 2+ (10 µM) (light blue line), re-oxidized rRNase I at 25 °C in the presence of Cu 2+ (0.3 µM) (green line), Cu 2+ (1 µM) (pink line), and Cu 2+ (10 µM) (grey line). All CD spectra were performed with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4, at 25 °C. ( D ) Intrinsic fluorescence emission spectra of native RNase and rRNase I at the end of re-oxidation process. Native RNase (black line), re-oxidized rRNase I at 37 °C (orange line), re-oxidized rRNase I at 37 °C with Cu 2+ (10 µM) (light blue line), re-oxidized rRNase I at 25 °C with Cu 2+ (0.3 µM) (green line), Cu 2+ (1 µM) (pink line), Cu 2+ (10 µM) (grey line). All the fluorescence emission spectra were recorded with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4, 25 °C. Experiments of re-oxidation of rRNases.
a With β-ME 11 µM. For sake of simplicity in the present work, we will continue to use the term auto-oxidation or spontaneous oxidation (used by Anfinsen and many other authors and textbooks) instead of the more correct “metal catalyzed oxidation”. The CD spectrum of the partially active and fully re-oxidized rRNase I at pH 8.5 with or without Cu 2+ did not overlap the one of the native enzyme, indicating lower amounts of beta sheets and turns ( Figure 1 C and Table 2 ). The intrinsic fluorescence of the six tyrosines of rRNase I gave further insights. In fact, the spectrum of the re-oxidized protein does not overlap the one of the native enzyme, despite all disulfides were formed. As it will be discussed below, this is a signal that tyrosines, in the re-oxidized rRNase I, reside in an increased non-polar environment compared to the native enzyme ( Figure 1 D) [ 14 ]. This indicates different tertiary structures formed as a consequence of non-native disulfides. Interestingly, the presence of sub-stoichiometric amount of β-ME (11 μM against 14 μM of rRNase I) allows a relevant recovery of activity (82%) in the range of that found by Anfinsen (80–100%) ( Figure 1 A and Table 1 ). Secondary structure analysis of the CD spectra of re-oxidized rRNase I.
As Anfinsen, after the reduction step, removed β-ME and urea using a Sephadex G-25 column equilibrated with acetic acid (pH 3.5), we also replicated this procedure obtaining a new reduced RNase (rRNase II, see Materials and Methods). The full oxidation of this reduced protein is again slow (4.5 day at 25 °C) ( Table 1 ) and CD analyses suggest a secondary structure only approaching that found in the native enzyme, showing a lower amount of beta structures ( Figure 2 A and Table 3 ). The complete oxidized enzyme also displays 49% of the native RNase activity, which represents the maximum recovery reached in all our experiments without a classical reshuffling mixture (GSH/GSSG) or without a sub-stoichiometric amount of β-ME ( Figure 2 B and Table 1 ). The intrinsic fluorescence spectra also indicate that oxidized protein acquired a tertiary structure different compared to the native RNase with the tyrosines inserted in a more hydrophobic environment ( Figure 2 C). This represents a further indication that incorrect disulfides oppose a complete re-formation of native secondary and tertiary structures of this enzyme.  Analyses at different stages of rRNase II (14 µM) during re-oxidation process in 20 mM Tris-HCl pH 8.5 under different conditions. ( A ) CD spectra of rRNase II at the end of the re-oxidation process. Native RNase (black line), re-oxidized rRNase II at 37 °C (light blue line), and re-oxidized rRNase II at 25 °C (navy blue line). All CD spectra were performed with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4, at 25 °C. ( B ) Recovery of activity of re-oxidized rRNase II at 37 °C (light blue column), at 25 °C (navy blue column), and residual activity of re-oxidized rRNase II 2.0 µM at 30 °C in 50 mM sodium phosphate buffer pH 7.5 with GSH/GSSG (2 mM/0.4 mM) and 5 mM EDTA (grey column). All the activities are compared to the activity of the native protein (black column). Activities were measured as described under Materials and Methods. The error bars represent the S.D. derived from three independent experiments. ( C ) Intrinsic fluorescence emission spectra of native RNase (black line), rRNase II (orange dotted line), re-oxidized rRNase II at 37 °C (light blue line), re-oxidized rRNase II at 25 °C (navy blue line), and re-oxidized rRNase II at 30 °C in 50 mM sodium phosphate buffer pH 7.5 with GSH/GSSG (2 mM/0.4 mM) and 5 mM EDTA (grey line). All the fluorescence emission spectra were recorded with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4, 25 °C. Secondary structure analysis of the CD spectra of re-oxidized rRNase II.
Furthermore, the replica of a famous experiment of re-oxidation of rRNase II at extreme low concentrations (1.8 µM), capable of recovering 100% of the original activity in 1 h, as described by Anfinsen [ 7 ], gave disappointing results. The full re-oxidation of rRNase II requires three days ( Table 1 ), which must be compared to one hour described by Anfinsen [ 7 ]. Moreover, the recovery of activity is only of 23% ( Table 1 ). The results claimed by Anfinsen for a spontaneous complete recovery of activity generated the belief that the reduced RNase spontaneously forms all its native structure and that proper disulfides can be formed later as stabilizers. This is what is described in many textbooks [ 1 , 2 , 3 ]. However, this is disputed by evidence that insufficient recovery of the original structure and activity can be obtained, likely due to the formation of incorrect disulfides. In fact, more recently, a quasi-stochastic mechanism for its oxidative folding has been proposed [ 15 , 16 ]. In addition, more recent studies agree with our results, observing that incomplete native structure can be recovered using a particular oxidizing agent [ 17 ]. Actually, we obtained a full recovery of the native activity and an emission spectrum close to the native one only after incubation with a reshuffling solution of GSH/GSSG ( Figure 2 B,C). This agrees with later studies which observed that the use of a mixture of reduced/oxidized DTT may contribute to restore the native conformation [ 18 , 19 ]. As above reported, also traces of β-ME ( Table 1 ) can produce very high recovery of the original activity. 2.2. Reduction of RNase under Different ConditionsThe recovery of activity, obtained using different reduction procedures, prompted us to investigate if different reductive conditions may generate structurally different rRNases (see Materials and Methods). CD spectra suggest that rRNase I and rRNase II display not negligible differences in secondary structure possibly responsible for the observed different restoration of activity ( Figure 3 A). For example, rRNase I shows a lower amount of beta structures compared to the rRNase II ( Table 4 ).  Spectroscopic analysis of the three rRNase I, rRNase II and rRNase III. ( A ) CD spectra of native RNase (black line), rRNase I (red line), rRNase II (orange line), and rRNase III (brown line). ( B ) Intrinsic fluorescence emission spectra of native RNase (black line), rRNase I (red line), rRNase II (orange line), and rRNase III (brown line). The CD spectra and the fluorescence emission spectra were recorded with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4 at 25 °C. Secondary structure analysis of the CD spectra of rRNases.
The rRNase I displays a much more fluorescence emission compared to the rRNase II confirming also different tertiary structures ( Figure 3 B). 2.3. RNase Reduction in the Absence of Denaturing AgentAnfinsen used 8 M urea and a relevant amount of β-ME (about 0.6 M) to reduce all the four disulfides in RNase [ 9 ]. The presence of urea was necessary because, in its absence, only a partially reduced RNase can be obtained, even after long incubation times at pH 8.5 [ 12 ]. In this way, however, nobody could distinguish between the effect due to urea and that due to the disulfide breakdown on activity and structure. The use of a stronger reducing agent such as DTT allows us to explore what happens to the enzyme activity and structure when the disulfides are progressively broken in the absence of a denaturing solution. DTT is a reagent of particular interest as its oxidized form is greatly stabilized being a cyclic disulfide, and it cannot form mixed disulfides when it interacts with protein cysteines. The incubation of native RNase with DTT causes a progressive and complete reduction of the four disulfides even in the absence of urea ( Figure 4 A). During this process, a relevant increase in the emission fluorescence of tyrosines has been found probably due to a shift in these residues toward a more hydrophobic environment and then to a change in the tertiary structure ( Figure 4 B) [ 20 ].  Analyses at different stages of RNase reduction. ( A ) Time-dependent formation of -SH per mole of rRNase III under reducing conditions (see Materials and Methods). The error bars represent the S.D. derived from three independent experiments. ( B ) Intrinsic fluorescence emission spectra of native RNase (black line), rRNase III (orange line), rRNase III with 6.0 -SH/mole (green line), 4.2 -SH/mole (blue line), and 1.9 -SH/mole (red line). All the fluorescence emission spectra were recorded with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4, 25 °C. ( C ) CD spectra of native RNase (black line), rRNase III (orange line), rRNase III with 6.0 -SH/mole (green line), 4.2 -SH/mole (blue line), and 1.9 -SH/mole (red line). All the CD spectra were recorded with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4, 25 °C. ( D ) Residual activity of rRNase during reduction pathway: native RNase (black column), rRNase III with 1.9 -SH/mole (red column), 4.2 -SH/mole (blue column), 6.0 -SH/mole (green column), and rRNase III (7.5 -SH/mole, orange column). All the experiments were performed as described under Materials and Methods. The error bars represent the S.D. derived from three independent experiments. A comparison of the CD spectra of the rRNase I and II, obtained in the presence of urea, with rRNase III, obtained in absence of urea (see Materials and Methods), suggests very similar reduced structures showing a relevant lack of secondary structures more evident for beta-sheets compared to the native form ( Figure 3 A and Table 4 ). Therefore, the presence of urea is not essential for the modification of the secondary structure, only determined by breakage of disulfides ( Figure 3 A and Figure 4 C). Surprisingly, the reduction of only one disulfide is able to generate some not negligible structural changes by lowering the amount of beta structures and reducing the activity to 74% ( Figure 4 C,D and Table 5 ). Other prominent structural and activity perturbations occur when other disulfides are broken step by step ( Figure 4 C,D and Table 5 ). Secondary structure analysis of the CD spectra of the reduction pathway.
A further indication that the breaking of just one disulfide is responsible for important structural changes comes from mass spectrometry data. When the reduction process with DTT was stopped when only one disulfide was broken, the partially reduced protein was incubated with bromopyruvate, a reagent that alkylates reduced cysteines in a few seconds. Digestion and ESI mass analysis of this modified protein revealed a quantitatively relevant amount of one peptide with an experimental [M + H] +1 monisot. = 2401.166 m/z , corresponding to the fragment 40–61 of RNase (CKPVNTFVHESLADVQAVCSQK) with a non-native disulfide bridge, i.e., Cys40-Cys58 (theoretical [M + H] +1 monisot. = 2401.164 m/z ) ( Figure 5 A–C). Both these cysteines are very distant in the native protein and linked with Cys95 and Cys110, respectively ( Figure 5 D).  MS and MS/MS analysis. ( A ) nano-HPLC-ESI-MS profile of the tryptic digest of the partially reduced rRNase III with 1.9 −SH/mole (1.25 μM). TIC: total ion current; NL: normalization level. ( B ) ESI spectrum of the chromatographic peak eluting in the 17.64–18.00 min retention time (RT) interval, resulting from the average (AV) of 12 ESI spectra. ( C ) Deconvolution of the ESI spectra reported in panel ( B ). The experimental [M + H] +1 monoisot. = 2401.165 m/z is very close to the theoretical [M + H] +1 monoisot. = 2401.164 m/z of the tryptic fragment 40–61 of RNase (CKPVNTFVHESLADVQAVCSQK) with a non-native disulfide bridge between Cys40-Cys58. The poor MS/MS spectra deriving from the HCD fragmentation of this connected peptide did not allow the confirmation of its structure, neither the other poor HCD MS/MS spectra permitted the characterization of the structure of other not-identified tryptic fragments deriving from a highly connected molten globule eluting in the initial and final part of the nano-HPLC-ESI-MS profile of panel ( A ) at different reduction ratios. ( D ) Three-dimensional structure of native RNase from bovine pancreas is represented in blue ribbons; cysteines are in ball and stick, the Cys40 and Cys58 are displayed in blue and yellow spheres. This finding is a strong indication that one of the two free cysteines, originated by DTT reduction of a single disulfide, interchanges with another distant disulfide and this becomes possible only if relevant secondary and tertiary structural changes occurred. Only a few experimental studies about the partial reduction of RNase have been performed, but they support our results. In particular, the study of Li and co-workers [ 21 ] identified the Cys40-Cys95 as one of the two disulfides first broken upon selective reduction. Even the theoretical study of Krupa and co-workers identified Cys40-Cys95 as the more susceptible disulfide to be the first cleaved [ 22 ]. 2.4. Refolding of rRNase Avoiding Disulfide FormationA further experiment, never done in the past, was designed to observe the evolution of the structure and activity of the reduced RNase, when incubated avoiding the disulfide formation (rRNase IV, see Materials and Methods). The rRNase IV was incubated at pH 7.4 in the presence of 0.2 mM DTT. Either after one hour or after 24 h, no appreciable recovery of activity was observed. CD spectra also indicate a relevant absence of alpha structures (~−70%), and an evident but not dramatic lack of beta structures (~−30%) ( Figure 6 A,B and Table 6 ).  Analyses of rRNase IV incubated under reducing conditions at 37 °C. ( A ) CD spectra of native RNase incubated in urea for 1 h without DTT (grey line), rRNase IV incubated in 0.05 M urea, 0.2 mM DTT and 1 mM EDTA for 1 h (emerald line), and rRNase IV incubated in 0.05 M urea, 0.2 mM DTT and 1 mM EDTA for 24 h (pink line). The CD spectra were recorded with a protein concentration of 1.25 µM in 10 mM sodium phosphate buffer pH 7.4 at 25 °C (see Materials and Methods). ( B ) Activity of rRNase IV incubated as in panel ( A ) and activity of native RNase (black column). All the experiments were performed as described under Materials and Methods. The error bars represent the S.D. derived from three independent experiments. Secondary structure analysis of the CD spectra of rRNase IV.
We believe this result to be important as, for the first time, it suggests that without disulfides a reduced RNase is completely unable to re-form native-like secondary structures. 2.5. The Effect of Urea on the Activity of the Native RNaseIn 1955, Anfinsen described a curious behavior of RNase. Apparently, prolonged incubation of this enzyme in 8 M urea at 5 °C did not produce any loss of activity [ 23 ]. This result was not disproved or modified later but only corrected in its interpretation. In fact, in 1989, Anfinsen, commenting his own work, says that it represents “ a beautiful example of how an entirely acceptable conclusion can be reached that is entirely wrong because of the paucity of knowledge at that particular time ” [ 24 ] (pp. 197–198). This refers to his previous (wrong) conclusion that an ordered shape of a protein is not strictly needed for its catalytic function if the structure of the active site is intact. In fact, further studies revealed that some apparent increment of activity occurs at high urea concentration probably due to an increased solubility of the reaction product or to a denaturation of the RNA thereby making it more available to the digestion by the enzyme [ 25 ]. In reality, the RNase activity is slightly lowered at similar urea concentration [ 25 ]. Thus, the full preservation of the RNase activity in 8 M urea, as reported by Anfinsen, is certainly a curious result, and my co-workers and I have been interested in replicating this experiment. However, as shown in Figure 7 A the presence of 8 M urea strongly inhibits RNase either after short or long time of incubation. Our experiments also confirmed an apparent increase in activity at lower urea concentrations (up to 6 M) and a slight decrease in activity at 7 M urea ( Figure 7 B). Thus, the lack of inhibition due to 8 M urea found by Anfinsen and co-workers [ 23 ] was possibly due to some overestimation of the urea concentration occurred in their activity measurements.  Activity of native RNase (2 µM) with different urea concentrations. ( A ) Residual activity of native RNase incubated for different times in 8 M urea at different temperatures: 70 h at 5 °C (green column), 10 min. at 25 °C (orange column), 2 h at 25 °C (red column), and 70 h at 25 °C (brown column). The activity of the native protein is also reported (black column). The experiments were performed as described under Materials and Methods. ( B ) Normalized activity of native RNase incubated for 1 h at 25 °C under different urea concentrations from 1 to 8 M urea (six shades of blue columns). All the activities are compared to the activity of the native protein (black column). The experiments were performed as described under Materials and Methods. The error bars represent the S.D. derived from three independent experiments. 3. DiscussionAlthough during the past sixty-five years a lot of studies have been published about the oxidative folding and reductive unfolding of RNase, data present in this paper allow us to update some details not previously underlined. First of all, the always-cited Anfinsen’s experiment of a spontaneous complete activity recovery of RNase by means of a simple exposition of the reduced enzyme to air oxygen [ 4 , 26 ] cannot be easily reproduced: no more than 30–50% of the original activity was obtained under variable conditions of pH, enzyme concentrations and reduced RNase preparations. Thus, the correct folding does not appear so straight and simple process. The presence of a reshuffling mixture is necessary to reach around 100% of a native activity ( Figure 2 B), as also noted by other researchers [ 13 , 20 , 27 ]. We speculate that the complete restoration of activity, as observed by Anfinsen, was probably due to traces of residual β-ME in the reduced enzyme sample. Such possibility is not unlikely seeing the not complete separation of reduced RNase from β-ME in the G-25 column, reported in Figure 1 in the study by Anfinsen and Haber [ 9 ]. If present, this contamination may promote a reshuffling of the uncorrected disulfides. The almost complete recovery of activity in the presence of traces of β-ME as obtained by us and shown in Figure 1 A is a good support for this hypothesis. Alternatively, some activity overestimation could be another factor due to the complexity of the enzymatic assay used in the past [ 28 ]. Incidentally, the statement reported in many textbooks that Anfinsen removed denaturing and reducing agents by means of dialysis has no confirmation in the literature [ 4 , 6 , 7 , 8 , 9 , 29 , 30 ]. Data concerning the secondary structure, obtained by CD analysis during the spontaneous oxidation pathway, allow us to obtain important structural information not acquired in the past on the un-modified RNase. In parallel to the observed partial recovery of native activity, also the CD spectrum differs significantly from that of the native enzyme. More in detail, when all disulfide are reformed, a lack of beta structures is evident in the totally re-oxidized rRNase I and II ( Table 2 and Table 3 ). Changes occurring in the secondary structures during the reductive pathway in the absence of urea fulfilled other important information. All structural and catalytic properties of RNase reduced under mild conditions of temperature and pH (rRNase III) indicate that this enzyme displays a different secondary structure with a lower amount of beta structures and a progressive loss of activity ( Figure 4 C,D and Table 5 ). In particular, the simple reduction of a single disulfide is able to trigger relevant structural changes as suggested by the formation of the non-native Cys40-Cys58 disulfide, both residues very distant in the native structure ( Figure 5 A–D). Similarly, incubating the rRNase IV at physiological pH and temperature values under constant reducing conditions, the enzyme does not recover its original secondary structures and shows also a negligible enzyme activity ( Figure 6 A,B and Table 6 ). Therefore, the reduced enzyme is unable to fold back into a native conformation except by allowing it to reform its natural disulfides. In conclusion, the well accepted assumption that RNase refolds spontaneously into correct secondary and tertiary structures and that disulfides consolidate such natural architecture must be completely refused. An opposite scenario it seems more reasonable, i.e., that the native disulfide formation is the necessary requirement to assemble the polypeptide into complete and correct alpha and beta structures. Refined CD analyses, not disposable sixty years ago, advise for this conclusion. Obviously, CD spectra do not fulfill any information on the tertiary structure, but some indications can be obtained by the changes of the fluorescence emission of the six tyrosines present in RNase. For example, the relevant increase in fluorescence emission at 300 nm (compared to the one of the native enzyme) observed in all the re-oxidized RNases under different conditions, points for different tertiary structures beside the above described different secondary structure. More precisely, it appears that in these non-native structures, tyrosines are internalized in a more hydrophobic environment compared to the native enzyme [ 20 ]. Alternatively, a greater distance of these tyrosines from the disulfides, known as fluorescence quenchers, could explain these results, but again, this would confirm tertiary structure modifications [ 14 ]. In fact, the increase in fluorescence observed in all reduced enzyme forms cannot be simply due to the cleavage of disulfide because a relevant increase in fluorescence is observed when uncorrected disulfides are formed ( Figure 2 C). The reshuffling procedure, necessary for a complete recovery of activity indicates that some uncorrected disulfides are first formed with only partial development of secondary structures. The continuous breaking and reformation of these disulfides, promoted by catalytic amount of reducing agent, or by a mixture of GSH/GSSG, is the mechanism needed for the formation of all correct disulfides. This event is crucial and precedes the definitive folding into all secondary and correct tertiary structures [ 15 ]. The thermodynamic profile toward the native structure can be now schematized, where, in the most favorable condition, only about 50% of RNase recovers spontaneously its native conformation and correct disulfides, while a second 50% falls into an un-proper energy hole characterized by incomplete secondary structure, improper disulfide and very low activity ( Figure 8 ). A reshuffling procedure is necessary to convert it into the native conformation with natural disulfides. Assuming correct this mechanism, only four steps of reshuffling are needed to reach 94% of the native activity. This occurs only using rRNase II as lower levels of native conformations (20–30%) are obtained with rRNase I or rRNase III.  Putative energetic profile of RNase oxidative refolding pathways. From the totally reduced protein to native conformations through kinetic traps (incorrect disulfides) with reshuffling mixture (e.g., GSH/GSSG); without reshuffling mixture, it will not be possible to reach native conformations (left panel). From totally reduced protein to native conformations through intermediates (correct disulfides) (right panel). These schemes derived from the best experimental conditions (described in this study) of reduction and re-oxidation (i.e., rRNase II). The percentage of 50% is referred to the productive conformations that evolved to the native state and is derived from the enzymatic activities as reported in Figure 2 B. In conclusion, this study describes new details of the in vitro oxidative pathway described many years ago by Anfinsen, but we are aware that the in vivo process can proceed in different ways. The recent discovery of an ultra-rapid glutathionylation of Cys95 [ 31 , 32 ] when reduced RNase is in the state of molten globule and similar phenomenon found in other proteins (i.e., serum albumin, lysozyme, chymotrypsinogen and trypsinogen) [ 33 , 34 , 35 , 36 ] are an intriguing indication in that direction. 4. Materials and Methods4.1. chemicals and reagents. Ribonuclease A (RNase) from bovine pancreas (Type XII-A, 75–125 Kunitz units/mg protein), bromopyruvic acid, copper sulfate, dithiothreitol (DTT), ethylendiamminotetreaacetic acid (EDTA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), L-glutathione (GSH), oxidized glutathione (GSSG), β-mercaptoethanol (β-ME), ribonuclease A activity kit and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The GSH solutions were prepared immediately before use and the GSSG concentration was less than 1% as assayed by standard analytical procedures. All organic solvents were of LC-MS grade. Acetonitrile (ACN), methanol (MeOH), formic acid (FA) and water were from Merck (Darmstadt, Germany). Trypsin (Gold MS Grade) was from Promega (Madison, WI, USA). 4.2. Protein ReductionRNase concentration was evaluated by an extinction coefficient of 9440 M −1 cm −1 at 280 nm [ 37 ]. RNase reduction was carried out with a protein concentration of 0.13 mM in 10 mM sodium phosphate buffer pH 7.4, 8 M urea, 1 mM EDTA and 10 mM DTT at 37 °C for 120 min. The reduced protein was passed through Sephadex G-25 column (1 × 20 cm) equilibrated in 10 mM sodium phosphate buffer pH 7.4 at 25 °C (through the manuscript the reduced protein after this process is indicated as rRNase I), or G-25 equilibrated in 0.1 M acetic acid at 25 °C (through the manuscript the reduced protein after this process is indicated as rRNase II). The number of -SH/mole of the eluted protein was titrated with DTNB at pH 8.0 (ε M TNBS − = 14,100 M −1 cm −1 ), 25 °C [ 38 ]. Alternatively, reduction of RNase 0.1 mM (or 0.2 mM), in absence of denaturant, was carried out in 0.1 M sodium phosphate buffer pH 7.4, 1 mM EDTA and 100 mM DTT at 37 °C for different times. After reduction, the excess of DTT was removed by passing through a Sephadex G-25 column (1 × 20 cm) equilibrated with 10 mM sodium phosphate buffer pH 7.4 at 25 °C (through the manuscript, the reduced protein after this process is indicated as rRNase III). The number of -SH/mole of the eluted protein was titrated with DTNB at pH 8.0 (ε M TNBS − = 14,100 M −1 cm −1 ), 25 °C. The preparation of rRNase IV for the characterization of conformational changes in the presence of the reducing agent DTT was performed as above described (starting from a protein concentration of 0.3 mM) for the preparation of rRNase I omitting the Sephadex G-25 chromatography. The rRNase IV was diluted to a final concentration of 2 µM in 10 mM sodium phosphate buffer pH 7.4, 1 mM EDTA, 0.2 mM DTT and 0.05 M urea. The solution was incubated at 37 °C for 1 and 24 h before the CD analyses and enzyme activity assays. 4.3. Re-Oxidation of rRNaseThe re-oxidation of rRNase I, rRNase II and rRNase III was carried out in sixteen different manners: (i). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C; (ii). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (iii). rRNase I (9.2 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu 2+ 10 µM at 37 °C; (iv). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu 2+ 0.3 µM at 25 °C; (v). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, 11 µM β-ME, and Cu 2+ 0.3 µM at 25 °C; (vi). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu 2+ 1 µM at 25 °C; (vii). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu 2+ 10 µM at 25 °C; (viii). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C; (ix). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (x). rRNase II (1.8 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C; (xi). rRNase II (1.8 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (xii). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu 2+ 0.3 µM at 25 °C; (xiii). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu 2+ 1 µM at 25 °C; (xiv). rRNase III (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (xv). rRNase III (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C. At different times, the number of -SH/mole were determined using DTNB as titrant, and aliquots were taken to perform CD, fluorescence and activities analyses. Finally, (xvi). the oxidation of rRNase II using the mixture GSH/GSSG was performed according to the following procedure: rRNase II (2 µM) was incubated with GSH (2 mM) and GSSG (0.4 mM) in 50 mM sodium phosphate buffer pH 7.5, 5 mM EDTA at 30 °C. At different times an aliquot was analyzed for enzymatic activity. 4.4. RNase Activity AssayActivities of native RNase, native RNase at different urea concentrations, rRNase I and II, rRNase III with different amount of disulfide bonds, rRNase IV with 1 mM EDTA, 0.2 mM DTT and 0.05 M urea, and re-oxidized rRNase (i-xvi as reported above) were assayed by the ribonuclease A detection kit (Sigma-Aldrich, St. Louis, MO, USA) which uses RNA as substrate [ 39 ]. The substrate RNA was essentially in a single strand conformation (double strand RNA ≈ 3%) as assayed spectrophotometrically after thermal denaturation. When necessary, the protein was diluted to a final concentration of 2.0 µM, in 50 mM sodium phosphate buffer pH 7.5, 5 mM EDTA. Only for the activity of the native protein at different urea concentrations, the protein was diluted to a final concentration of 2.0 µM, in 50 mM sodium acetate buffer pH 5.0 with different amounts of urea. An aliquot (RNase final concentration 80 nM) was then added in cuvette to a solution containing RNA (0.05% w / v ), and 0.2 mM EDTA in 50 mM sodium acetate buffer pH 5.0. Only for rRNase IV, the concentrations of EDTA, urea and DTT were 40 µM, 2 mM and 8 µM, respectively. For the measurements of native RNase in 6 M, 7 M, and 8 M urea, the RNA was dissolved in the reaction buffer with 6 M, 7 M, or 8 M urea, respectively. The recovered activity was monitored spectrophotometrically in continuous at 300 nm, 25 °C. The data were reported as percentage of the ratio between sample and native RNase. Only in the case of native RNase at different urea concentrations, data were reported as normalized absorbance ratio between sample and native RNase. 4.5. Circular Dichroism SpectroscopyCD spectra of native RNase, native RNase in 0.03 M urea, rRNase I, rRNase II, rRNase III, and rRNase IV in 0.03 M urea, 625 µM EDTA, 0.125 mM DTT were performed in 10 mM sodium phosphate buffer pH 7.4, at 25 °C, and in all cases with a protein concentration of 1.25 µM using a spectropolarimeter Jasco J-1500 (Easton, MD, USA). The setting panel was: slit 2 nm, sensibility 20 mdeg, resolution 0.5 nm, and range values 190–260 nm using a quartz cuvette of 0.5 cm light path. CD spectra of native RNase and rRNase in the presence of 0.03 M urea cannot be extended below 200 nm due to the interference of urea. CD spectra for all the samples of re-oxidized rRNase I and re-oxidized rRNase II were acquired in the same manner. The analyses of CD spectra were performed using DichroWeb [ 40 ] and BeStSel software [ 41 ]. 4.6. Fluorescence MeasurementsAll the fluorescence analyses of native, reduced RNases (I–III) and re-oxidized rRNases (I and II) were performed on a Fluoromax-4 Horiba spectrofluorometer with an asymmetric quartz cuvette of 1 × 0.4 cm path length at 25 °C. The fluorescence emission spectra of native, reduced RNases (I–III) and re-oxidized rRNases (I and II), in all cases 1.25 µM, were recorded in 10 mM sodium phosphate buffer pH 7.4 between 290 and 350 nm, using the following parameters: excitation wavelength of 275 nm and slits 5–8 nm. 4.7. Preparation of rRNase Samples for Mass-Spectrometry AnalysisThe rRNase III at three different times of reduction (with a content of 2, 4, and 6 -SH/mole rRNase), was diluted to a final concentration of 1.25 μM in 10 mM sodium phosphate buffer with 1 mM bromopyruvic acid that alkylates residual protein cysteines within 1–2 s. Then, the samples were lyophilized. The samples (50 μg each one) were submitted to an overnight digestion at 3 °C using a porcine trypsin gold (mass spectrometry grade, resistant to autolytic digestion) in 1:50 ( w / w ) ratio with respect to the protein content. Enzymatic digestion was stopped by addition of 1 μL of 100% FA, and then 1μg of total protein content per sample was used for MS analyses. 4.8. HPLC-ESI-MS/MS AnalysisNano-HPLC/nano-ESI-Orbitrap Elite analyses were performed on an UltiMate 3000 RSLCnano System coupled to an Orbitrap Elite MS detector with an EASY-Spray nano-ESI source (Thermo Fisher Scientific, Waltham, MA, USA). EASY-Spray column 15 cm × 50 μm ID, PepMap C18 (2 μm particles, 100 Å pore size) (Thermo Fisher Scientific), was used for bottom-up analyses, in coupling to an Acclaim PepMap 100 cartridge (C18, 5 μm, 100 Å, 300 μm i.d. × 5 mm) (Thermo Fisher Scientific). Bottom-up nano-HPLC-MS/MS analyses were performed using aqueous solution of FA (0.1%, v / v ) as eluent A and ACN/FA (99.9:0.1, v / v ) as eluent B in the following gradient elution: (a) 5% B (2 min), (b) from 5% to 60% B (30 min), (c) from 60% B to 99% (10 min), (d) 99% B (10 min), (e) from 99% to 5% B (2 min), and (f) 5% B (10 min) at a flow rate of 0.3 μL/min. The injection volume was 5 μL corresponding to 1 μg of total protein content per sample. Peptide trapping and concentration were obtained by loading the sample for 5 min into the Acclaim PepMap 100 nano-trap cartridge operating at 10 μL/min in eluent A. Chromatographic separations were performed at 35 °C. The Orbitrap Elite instrument was operating in positive ionization mode, performing MS/MS fragmentation by higher energy collisional dissociation (HCD) of the five most intense signals of each spectrum, measured at a 60,000 resolution in 150–2000 m / z acquisition range, in data-dependent scan (DDS) mode. The minimum signal was set to 500.0, the isolation width to 2 m / z , the default charge state to +2, and the activation Q to 0.25 MS/MS spectra acquisition was performed in the Orbitrap at 60,000 resolution. 4.9. Data and Graphical RepresentationThe experimental data reported in Figures and Tables were analyzed and expressed as Mean ± Standard Deviation (S.D.). Data were obtained from three independent experiments performed in different days by the same operators using the same instruments. The graphic and results visualization were obtained using GraphPad Prism software v5.0 (La Jolla, CA, USA). Crystal structure of native RNase is derived by PDB id: 1FS3 [ 42 ], the structure was drawn by UCSF Chimera [ 43 ]. AcknowledgmentsAuthors would like to thank Alessandra Boccaccini, Sara Bobone and Paolo Calligari for helpful discussion. Funding StatementThis research received no external funding. Author ContributionsConceptualization, A.B.; data curation, G.G. and S.N.; formal analysis, G.G., S.N., D.C., F.I. and M.C.; investigation, G.G., S.N., D.C. and F.I.; methodology, A.B.; project administration, G.R.; resources, A.B.; supervision, G.R.; validation, M.C., and A.B.; visualization, G.G. and S.N.; writing—original draft preparation, G.R.; writing—review and editing, M.C., A.B. and G.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board StatementNot applicable. Informed Consent StatementData availability statement, conflicts of interest. The authors declare no conflict of interest. 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COMMENTS
The view that 3-D structure is determined by 1-D amino acid sequence, rested for three decades on a series of enormously influential experiments carried out by Christian Anfinsen, Frederick White, Michael Sela, and Edgar Haber at the National Institutes of Health. Behooves us to examine carefully those initial experiments.
Protein folding refers to the set of ordered pathways by which protein folds into their native functional confirmation. Protein folding is primarily driven by hydrophobic forces. Anfinsen's Experiment. First step.. The first insight to this question was provided by Christian Anfinsen at the NIH. He was working on the properties of ribonuclease ...
Before we study the classic experiment of protein folding conducted by Anfinsen, study the simpler analogy below: Figure: Socks and protein folding The classic experiment of Anfinsen has shown that, at least for some proteins, all the necessary and sufficient information required to direct the folding of a protein into the native state is ...
Anfinsen's Experiment of Protein Folding. In the 1950s, Christian Anfinsen conducted a series of experiments in which he determined that all the information needed to form the three-dimensional structure of the polypeptide is stored in the specific sequence of amino acids in that polypeptide. Later experiments confirmed this fact - that primary ...
Anfinsen wanted to know if positioning the right number of the right amino acids in the right sequence was enough to form a folded protein. For this work he used ribonuclease (RNase) from cows—obtained, interestingly enough, from a slaughterhouse. ... the protein automatically refolded into its active shape. This experiment demonstrated that ...
The classic experiment of Anfinsen showed that, at least for some proteins, all the necessary and sufficient information required to direct the folding of a protein into the native state is present in the primary sequence of a protein. ... The first step is really a disulfide shuffle, which, when coupled to subsequent steps, leads to de novo ...
In the fall of 1972, while working in a biochemistry laboratory as an undergraduate at Brown University, I heard about an astonishing experiment for which Christian Anfinsen was receiving the ...
An experiment that gave us a particular satisfaction in connection with the translation of information in the linear amino acid sequence into native con- ... C. B. Anfinsen 57 Fig. 2. Schematic representation of the reductive denaturation, in 8 molar urea solution containing 2-mercaptoethanol, of a disulfide-cross linked protein. The conversion ...
A schematic representation of Anfinsen's experiment. Full size image. In the first experiment, highly purified and active RNase A was treated with excess β-mercaptoethanol and 8 M urea. ... this step is always preceded by the formation of secondary structural elements. Research carried out in the 1980s and 1990s by several laboratories ...
Anfinsen's experiment and protein folding - This lecture explains about the protein folding experiment.http://shomusbiology.com/Download the study materials ...
The thiol group of Cys is most reactive of any amino acid side chain. HN - C - S-H C=0 It ionizes at slightly alkaline pH; the thiolate ion is reactive form; For free amino acid pka of 9.0-9.5. In Proteins pk 8.5; but pk very sensitive to local electronic environment, so cannot assume this within proteins.
For more information, log on to-http://shomusbiology.weebly.com/This video lecture explains the Anfinsen's experiment about protein folding and it also illus...
Anfinsen wanted to understand what the spatial organization, or 3D shape, of a protein depended on, essential for its correct functioning. Searching in his experiments for a single molecule whose activity was easily measurable, he opted for ribonuclease, an enzyme that breaks RNA into fragments. And to study it, he had an idea that no one had ...
Anfinsen Experiments. Description: This resource contains information regarding study materials. pdf. 607 kB. Anfinsen Experiments. Download File. DOWNLOAD. This resource contains information regarding study materials.
Folded, 3-D structure of ribonuclease A. Anfinsen's dogma, also known as the thermodynamic hypothesis, is a postulate in molecular biology.It states that, at least for a small globular protein in its standard physiological environment, the native structure is determined only by the protein's amino acid sequence. The dogma was championed by the Nobel Prize Laureate Christian B. Anfinsen from ...
Key Words: biology investigation; protein folding; Anfinsen. Introduction The study will try to adapt Anfinsen's Nobel-winning experiment about protein folding (Anfinsen & Haber, 1961) into biology investi-gation for secondary and college students. This experiment is signifi-cant for biology learning not only for its associations with some core
Anfinsen's coauthor on this JBC Classic also went on to make important contributions to science. Edgar Haber (1932-1997), who earned his A.B. (1952) and M.D. (1956) from Columbia University (1952), was a research associate at the Laboratory of Cellular Physiology when he published the paper with Anfinsen. He eventually became an eminent ...
The term 'Anfinsen cage' was proposed to summarise the idea that GroEL increases the yield of correctly folded protein by encapsulating each partially folded chain inside its oligomeric structure, where it can continue to fold as in the classic protein renaturing experiment pioneered by Anfinsen [10]. Figure 1A illustrates the basic mechanism suggested by the results of many ingenious in ...
Download scientific diagram | Scheme of Anfinsen's experiment. See text for details from publication: Protein Folding | Since Anfinsen's famous experiments in the 1960s, it has been known that the ...
Paper: Expanding Anfinsen's Principle: Contributions of Synonymous Codon Selection to Rational Protein Design.. In 1961, Anfinsen performed his now (in)famous denaturing experiment upon ribonuclease A, a small one hundred residue globular protein. He showed that it could both unfold and refold via the addition and subsequent removal of chemical substances.
These later steps are believed to resemble those occurring in the test tube in an Anfinsen refolding experiment — hence the term 'Anfinsen cage' is used to describe the structure responsible. It is not yet clear whether the final steps in folding to the native form occur inside or outside the cage: this may vary between proteins with ...
As Anfinsen, after the reduction step, removed β-ME and urea using a Sephadex G-25 column equilibrated with acetic acid (pH 3.5), we also replicated this procedure obtaining a new reduced RNase (rRNase II, see Materials and Methods). ... First of all, the always-cited Anfinsen's experiment of a spontaneous complete activity recovery of RNase ...