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Loop Fold Structure of Proteins: Resolution of Levinthal’s Paradox
Abstract According to Levinthal a protein chain of ordinary size would require enormous time to sort its conformational states before the final fold is reached. Experimentally observed time of folding suggests an estimate of the chain length for which the time would be sufficient. This estimate by order of magnitude fits to experimentally observed universal closed loop elements of globular proteins – 25-30 residues. Key words: Levinthal’s paradox, protein folding, chain conformation, closed loops, loop fold structure
Igor N. Berezovsky1,* Edward N. Trifonov2
of Structural Biology
The Weizmann Institute of Science P.O.B. 26 Rehovot 76100 Israel
Institute of Evolution University of Haifa Haifa 31905 In 1968 Levinthal in his report of which only brief summary is available (1) noted that reversibly denaturable proteins during transition from “random disordered state into a well-defined unique structure” have to go through conformational space with immense number of states, so that the time required for visiting all the states would also be very large. Indeed, (e.g. (2)) for a protein chain of length L = 150 (residues) with n = 3 alternative conformations for every residue, the time t required for sorting out all possible conformations of the chain is: t = nL ⋅ τ = 3150 ⋅ 10-12 s = 1048 yrs  Israel
(τ = 10-12 s is time for elementary transition (2)). Observed values of t are in the range 10-1 to 103 s (2), that is, the full sorting as above is impossible. Thus, protein folding has to proceed along a certain path that would avoid most of the conformational space. The path should somehow be directed by an as yet unknown sequence-dependent folding rule(s). The size of the short chain for which the observed time span of 10-1 to 103 s would be sufficient for trying every possible state can be calculated from , with the same assumptions, as l0 = lg(t/τ) = 23 to 31 residues. In this case all conformlg n ations could be tried during the given time, and the lowest energy state(s) attended. Being logarithmic this estimate is rather insensitive to the choice of the values for n which according to various authors may change between 1.6 and 10 elementary conformations (3, 4). With these extreme values the above estimate spans the range l0 = 11 to 74 residues. The l0 value may, thus, serve as a rough estimate of the size of the units (chains or chain segments), which could attend all conformational states during observed time. The estimated size of the hypothetical unit is identical to the optimum of loop closure for polypeptide chains, 20 to 50 residues (5), and to the observed size of recently discovered closed loop elements, 25-30 amino acid residues (5-7), of
Phone: 972-8-9343367 Fax: 972-8-9344136 E-mail: Igor.Berezovsky@weizmann.ac.il
6 Berezovsky and Trifonov
which globular proteins are universally built. One can hypothesize that the closed loops are also elementary folding units. In this case their linear arrangement within the protein folds (5, 8) would suggest a straightforward folding path: sequential formation of the closed loop units, along with their synthesis in the ribosome. If such successive formation of the stable folding units in the course of translation is assumed, it will require time proportional to the number of the units, that is, only several fold larger than required for a single unit. The above scenario is consistent with the typical rates of translation, 3 to 20 residues per second (9). Synthesis of the protein of length L = 150 takes, thus, 8 to 50 seconds, which is a fair match to the above range of folding rates. Thus, according to the estimates above the consecutive formation of the loop-like folding units of 25-30 residues is by the order of magnitude time-wise consistent with both folding and translational experiments. Acknowledgement We are grateful to Prof. A. Yu. Grosberg for valuable comments and enlightening discussions.
References and Footnotes 1. 2. 3. 4. 5. 6. 7. 8. 9. Levinthal, C. J. Chim. Phys. Chim. Biol. 65, 44-45 (1968). Branden, C. and Tooze, J. Introduction to Protein Structure, Garland Publishing (1999). Bryngelson, J. D. and Wolynes, P. G. Proc. Natl. Acad. Sci. USA 84, 7524-7528 (1987). Pande, V. S., Grosberg, A. Yu. and Tanaka, T. Reviews of Modern Physics 72, 259-314 (2000). Berezovsky, I. N., Grosberg A. Y. and Trifonov, E. N. FEBS Letters 466, 283-286 (2000). Berezovsky, I. N. and Trifonov, E. N. J. Biomol. Struct. Dyn. 19, 397-403 (2001). Berezovsky, I. N. and Trifonov, E. N. J. Mol. Biol. 307, 1419-1426 (2001). Berezovsky, I. N. and Trifonov, E. N. Prot. Engineering 14, 403-407 (2001). Varenne, S., Buc, J., Lloubes, R. and Lazdunski, C. J Mol Biol. 180, 549-576 (1984).
Date Received: July 6, 2002
Communicated by the Editor M. D. Frank-Kamenetskii
In principle. a nucleation mechanism can account for all these major features simultaneously and resolves the Levinthal paradox. i. and thus much more stable than the denatured state of protein chain.jbsdonline. Key words: protein folding. 3) in an attempt to resolve the Levinthal paradox (4). Issue Number 3. entering the tertiary protein fold in the already found form. Long life of the once found form means that it is thermodynamically stable as compared to the initial denatured state of the same piece of the chain. Specifically.e. rate of folding Alexei V. in a visible quantity. two-state kinetics..e. However. to explain how a problem of sampling the impossibly large number of conformations by the folding protein chain can be avoided. as building blocks at the next stage of folding. Pushchino Moscow Region. for the time comparable with that of its own formation. (2002) ©Adenine Press (2002) An Opinion Piece: Conversation on Levinthal Paradox & Protein Folding #1 Cunning Simplicity of a Hierarchical Folding http://www.Journal of Biomolecular Structure & Dynamics.ru 311 . This refers to proteins Phone/Fax: (+7-095) 924 0493 Email: afinkel@vega. However.protres. and then the bigger structures obtained at the second stage serve as the building blocks for the next stage. though. On the contrary. a hierarchic mechanism can help to avoid sampling of the huge conformational space. more stable than the sum of these sable folded units.. Such a mechanism may work only when the native structure is much more stable than the unfolded or denatured state of protein chain. of only the native and the unfolded molecules during folding of moderate size (single-domain) proteins. without any significant reconstruction. many single-domain proteins (having up to 200–250 residues) successfully fold near and even in the point of thermodynamic equilibrium between their native and denatured states. any hierarchic mechanism (including the one suggested by Berezovsky & Trifonov) implies that the formed folding unit has to preserve its once found form at least until the next unit will be formed.com Abstract A hierarchic scheme of protein folding does not solve the Levinthal paradox since it cannot provide a simultaneous explanation for major features observed for protein folding: (i) folding within non-astronomical time. i. mid-transition. Levinthal paradox. a high stability of the native structure is not obligatory for folding. when protein folding occurs near the point of thermodynamic equilibrium between the native and denatured states of the protein. As demonstrated by numerous chevron plots (5-8). Russia Berezovsky & Trifonov (1) have recently revisited an attractive idea of a hierarchic protein folding (2. folding nucleus. this means that the native protein is. Berezovsky & Trifonov in their clearly written paper (1) assume that local “closed loops” of 25-30 residues (the smallest folding units) find their lowest-energy structures by an exhaustive search of all their conformations and then stick together. etc. (ii) independence of the native structure on large variations in the folding rates of given protein under different conditions. and (iii) co-existence. A “hierarchic mechanism” means that some structures formed at the first stage serve. Since the folding units then stick together. I would like to note that any hierarchic scenario cannot serve as a general solution of the Levinthal paradox. Indeed. It cannot work. in turn. co-existence of the native and the unfolded phases. Finkelstein Institute of Protein Research Russian Academy of Sciences 142290. ISSN 0739-1102 Volume 20.
10). of only the native and the denatured protein molecules during the folding near the point of thermodynamic mid-transition between these two states. 14). . the hierarchic mechanism means that the native protein structure is controlled by kinetics rather than by stability of the whole protein. The same refers (19) to the simplest versions of a funnel model of protein folding (20-22). one has to explain why the same native protein structure results from foldings held under different conditions and having 1000-fold difference in speed (which. observed in some cases (15. that have detectable folding intermediates far from the equilibrium point). “semi-native” forms. crystallization) in macroscopic systems (9. it has been shown that the nucleation mechanism (that pays main attention to the boundary between the folded and denatured phases within a protein molecule) can resolve the Levinthal paradox (11) and leads to realistic estimates of the protein folding rates (12). Acknowledgements This work was supported by an International Research Scholar’s Award from the Howard Hughes Medical Institute and by a grant from the Russian Foundation for Basic Research.. In its strict form. Therefore.e. a hierarchic folding seems to take place in large multi-domain proteins whose denaturation is not an “all-or-none” transition but proceeds as a sum of “all-or-none” denaturations of their domains (18). though most of them have to have an enhanced stability for pure statistical reason (17). A hierarchic folding does not provide a general solution of the Levintal paradox since it cannot simultaneously explain for major features. in a visible quantity. as well as to those that have three-state folding kinetics (i. are unstable and therefore present in a very small quantity. it is noteworthy that this question does not arise at all when the native fold is determined by its stability. 16). Specifically.. It is worthwhile to note that the latter fact is easily explained if the choice of the native fold is determined by its stability (rather than by folding kinetics): it is not obligatory that every element of the lowest-energy fold has an enhanced stability. On the other hand. However. An “all-or-none” transition is a microscopic analog of first order phase transitions (i. i. The presented considerations give an important criterion of applicability of any protein-folding model to single-domain proteins: whether or not this model can explain protein folding near the point of thermodynamic mid-transition between the folded and unfolded states. (ii) independence of the native structure on large variations in the folding rates of given protein under different conditions. one has to explain why the folding with detectable folding intermediates (far from the mid-transition) is not drastically faster than the folding that does not have such intermediates. A hierarchic folding scheme discussed in this paper cannot satisfy this criterion. has to lead to folding units of rather different sizes). On the contrary.and renaturation of single-domain proteins (9). while others. Also. do not prevent protein from correct hierarchic folding. according to the logic of Berezovsky & Trifonov (1).312 Finkelstein having a simple two-state folding kinetics.. 13. The discovery of this fact in kinetics was actually a re-discovery of wellknown “all-or-none” thermodynamics of de. The only necessary prerequisite of such a transition is an energy gap between the lowest-energy native fold and misfolded structures (11. observed for folding of single-domain proteins: (i) folding within non-astronomical time.e. The “all-or-none” transition means that only the native and denatured proteins are present (close to the mid-transition) in a visible quantity. this does not mean that a hierarchic folding scenario is completely inconsistent with all proteins. and why the evidently non native-like intermediates. it is not a surprise that this transition in proteins was shown to follow a nucleation mechanism (8) that is well known (in physics) to be specific for the first order phase transitions (10). and (iii) co-existence. the simplicity of a hierarchic folding is cunning. the nucleation model of protein folding meets this criterion and resolves the Levinthal paradox. If so.e. Moreover.
. 24. M. USA 92. and Rose. Baldwin. A. V.. 8. A. D. Biomol. 19. Zwanzig. 18. 313 Cunning Simplicity of a Hierarchical Folding Date Received: September 16. 115-121 (1997). D. Bicout. 77-83 (1999). and Gutin. Serrano.. S. Chim. and Szabo. 13. Natl. Dokl. Baldwin. Privalov. P. Curr. 9029-9033 (1995). Proc.. T. 20-22 (1992). L. 3. Protein Science 9. C. 92-94 (2001).. R.. 8. 7524 (1987). and Dill K. B. P. N. Adv.. A. Landau. 9. Dyn. 16. Badretdinov. J. 9029-9033 (1992). B. Goldstein. Proc. M. Matouschek. Y. Wolynes.. Statistical Physics. Struct. Struct. 452-465 (2000). Folding & Design 2. Fersht. Bryngelson and P. 521-523 (2001). Proteins 30. Acad. Bogatyreva. Akad. Nature 340. and Trifonov. and Wolynes. Luthey-Schulten. USA 89. Privalov. R. Protein Engineering 14. 19. A. Opin. 167-241 (1979). 22. 65. Natl. D. 7. Acad. Sci. 735-737 (2002). A. 2002 Communicated by the Editor Ramaswamy H Sarma . Sci. A. A. Pergamon (1959). Adv. Biomol. Ya. Protein Chem.. N... Sci. 12. A. A.. J. J. E. L.. R. J. N. N. A.-I. Chan H. A.. D. 3-9 (1997).. Nauk SSSR 210. 15. 20. Biol. Dyn. Acad. Protein 23. Sci.. Finkelstein. Proc. 2. M.. L. 11. 10. 113-118 (2001).. S. 4. V. Kellis. Segawa. Fernandez. and Finkelstein. L. 1-104 (1982). Szabo. 2473-2488 (1984). Z. 35. and Sugihara. London. Berezovsky. and Lifshitz. 1213-1215 (1973). Finkelstein.. Natl. E. 26-33. USA 89 4918-4122. 21. S. Jr. R. and Badretdinov. J. G. Protein Chem.. V. Levinthal.. 142-150 (1995). ibid. Kiefhaber. V. Trends Biochem. Ptitsyn. and Bagchi. G.. V... and Finkelstein A. FEBS Letters 489. J. Galzitskaya. Acad. 2-33 (1998). 17. G. Struct. 14. USA 84. P. 44-45 (1968). B. Ivankov.. 7. and Fersht A. T. R. 20..References and Footnotes 1. 33. A. 122-126 (1989). A.. L. Chim. I. 5. A.. Biopolymers 23. Biol. 5-6 (2002). Proc. O. R. Nature Struct.. Biol. Sci. O. 6. Natl. Phys. L.
Dyn. its size and structure. due to long-living native and non-native meta-stable states (5. thus. Struct. become either smaller (e. The respective simple formalism provides a rough estimate of the size of such a chain. The same calculation in reverse as in (2) leads to what we would call the Levinthal limit – in other words the size of the polypeptide chain or part thereof for which the observed times of protein folding would be sufficient to sort out all possible conformations. due to Phone: 972-8-9343367 Fax: 972-8-9344136 Email: Igor. This initiated decades of theoretical and experimental research (16-20) to figure out what is a time-wise agreeable path of folding of the full-size proteins.ac. and every contribution towards their characterization (9-13) is important. Struct. Finkelstein (1).com Entia non sunt multiplicanda praeter necessitatem (Occam) Abstract In response to the criticism by A. Berezovsky1. Trifonov2 1Department of Structural Biology The Weizmann Institute of Science P.O. Israel The main points of our Communication (2) – the structural units of folding and closed loops as likely candidates for that role – are not challenged by A. Dyn. Biomol. It may. Issue Number 3.Berezovsky@weizmann. Key words: Levinthal paradox. under the assumption that there are no returns to the same conformations during the sorting. or larger (e. apparently. protein folding. biological relevance.jbsdonline. 26. More elaborate approaches should take into consideration both physical factors and biological circumstances that may influence the estimate. 2002) several issues are dealt with. and the structural component to it . Our study suggests a rather narrow size range for the hypothetical folding units. Biomol. 20. The criticism (J. But his piece rather invites to a separate chapter in protein folding studies – a hierachical folding (3-8). but rather a simple illustration of how the elementary structural units may partake in the initial stages of folding process during translation. 2002) on the hierarchical protein folding is also briefly addressed. Rehovot 76100. 20. folding units. Biomol. 5-6. Igor N. ISSN 0739-1102 Volume 20. 311-314. But again. This biological dimension is. and the necessity of further characterization of the units for the elucidation of the protein folding in vivo are discussed.chain-return nature of the units (14). Israel 2Genome Diversity Center Institute of Evolution University of Haifa Haifa 31905. The passage in our note (2) “If such successive formation of the stable folding units in the course of translation is assumed. and it is not about hierarchical folding. Importance of the notion of elementary folding unit.7)). and their fate during protein folding may follow many different scenaria (6. (2002) ©Adenine Press (2002) An Opinion Piece: Conversation on Levinthal Paradox & Protein Folding #2 Back to Units of Protein Folding http://www.* Edward N.B. Calculations for the ordinary protein sizes on the basis of Levinthal’s original idea (15) lead to astronomical times. Dyn.il 315 . Struct. 7). 311-314. 20. along with the synthesis of the polypeptide chain. 2002) of our Communication (J.g. a crucial component in protein folding problem. The very notion of independently folding units is an important concept.Journal of Biomolecular Structure & Dynamics. The structural units of folding may or may not remain intact once formed.g. the discussion on these scenaria is well beyond the scope of our original Communication (2). …” is not a statement. Finkelstein (J.
M. 735-737 (2002). Biomol. I. 22. A. Gutin. Biol. Levinthal... USA 93. Kirzhner. Gething. Sci. V. I. L.. and Daggett. 25-30 amino acid residues. Panchenko.. Fersht. N.. Berezovsky. Luthey-Schulten. Fersht.. E. that indicate the same size range. in press (2002).. Shakhnovich. as the one suggested by the polymer statistics of the polypeptide chains (14) and by the observed rates of translation (2). 26)). 21. Galzitskaya. A. Trends Biochem. N. and Trifonov. I. 12. A. Acad. Proc. 557-565 (1997). Berezovskii. R. and Bedows. V.. 7. 24.. and Sambrook.. Cell 108. A.. 53. Mol.. N. Evol. S. Natl. 814-817 (1999). Sci. Dyn. 1213-1215 (1973). 24). I. Dyn. E. FEBS Letters 466. Struct. Chem. L. A. Acad.. V. 26. Nature 355. Yu. P. A. G. and Wolynes. Biol. 6. P. We believe. References and Footnotes 1.. Acad. N. 21-24). 113-118 (2001). Struct. Proc. A. 23. L. and Tanaka. J. N. Acad. that both experimental and theoretical studies on the units of folding is the right way to elucidation of how exactly the protein folds in vivo. I. R. Sci. I. The balance is still to be found. R. J. 26-33 (1999). B. M. S. and Trifonov.316 Berezovsky and Trifonov special sequence organization (4. Phys. N. 259-314 (2000). 24. Cole. Natl. 272. Shakhnovich. and Trifonov. Biol. G. 7. USA 90. N. E. Ivankov. I.... O. Biol. V. Nauk SSSR 210. Biol. Ptitsyn. N. Panchenko. 311-314 (2002). Finkelstein. R. Curr. 7. Sci. 5. 814-817 (1999). V. 283-286 (2000). Biol. and Tumanyan. Chim. 2002 Communicated by the Editor Ramaswamy H Sarma . Possible clues are apparent evolutionary fingerprints in protein sequences (23. R. D. 12972-12975 (1994). Nat. Comparative and Functional Genomics. 13. Opin. Yu. N. W. Trifonov. 10026-10036 (1994). 25. E. 1525-1529 (2000). 33-45 (1992). Nat. 15. J. V. Pande. 2. and Gutin. T. Shakhnovich. Berezovsky. E. A. Abkevich. and Wolynes.. V.. Struct. and possible structural guidance within the ribosome or in chaperones (25. 8. G. V. Natl. M. N. Struct. Esipova. Date Received: October 25. 9. Struct. 6. Reviews of Modern Physics 72... 272. J. 394401 (2001). E. 3-9 (1997). Z... and Rose. Y. R.. and Rose. Luthey-Schulten. G. Chim. Ruddon. 6.. Grosberg.. Grosberg A. V. Pande. 10. A. J. G. A. Sci. 44-45 (1968). J. Struct.. 24. J. N. 95105 (1997). R.. 20. E. A. 16.. 2008-2013 (1996).. 19. 18. Dyn. O. I. 7524-7528 (1993). 20. T. Trends Biochem. Biomol. Biol. USA 97.. C. Berezovsky. Dokl. Baldwin. R. Struct. Akad. 4. Biomol. Biochemistry 33. Curr. FEBS Lett 489. 573-582 (2002). D.. L. 5-6 (2002). J. and Berezovsky. and Tanaka. Opin. and Finkelstein A. Proc. 65. A. Mol. 3125-3128 (1997). R. Fersht. Baldwin. Natl. J. 19. 17.. Sci. 11. Baldwin. Biophysics 42. D. 77-83 (1999). Fernandez.. Baldwin. I. E. 3.. Kirzhner. 14. Grosberg. USA 91. 20. R. M.. G. Proc. Z. R. 29-40 (1997)..
even if they were to complete the exhaustive testing. Trifonov (IBET for brevity). The opinion is formulated that the discussions of Levinthal paradox should now fly to the new spheres.com Abstract We estimate that the longest protein chain capable of exhaustive sampling of all its conformations within a millisecond is shorter than 15 residues. where M is the number of distinct conformations. and the real time was much longer than that. however. they found a large plaza with dozens of pavilions. Assuming visit to one pavilion takes time τ. Fraunfelder (3) suggests to Email: grosberg@physics. H. When biophysicists arrived.jbsdonline. one could have naively expected that after time close to Mτ the testing would be over. with each individual in the cloud undergoing random walks. In fact. The site was a rural place in the center of a famous wine producing region.umn. USA 2Institute of Biochemical Physics Russian Academy of Sciences Moscow 117977. particularly in the ones initiated by the recent very clearly written article (2) by I. But there is also another more interesting reason: the time necessary for the random walk to visit all M sites does not scale as Mτ. such that reliable folding does not require exhaustive conformation sampling.Journal of Biomolecular Structure & Dynamics. Before long. M is so large that. because M is astronomically large. the cloud of biophysicists seemed perfectly obeying the diffusion equation. it can be significantly larger than that. ISSN 0739-1102 Volume 20. a large biophysics meeting was held in the then Soviet Republic of Georgia. namely. One possible reason is trivial: wine testers. Such expectation was proved to be terribly wrong. each representing a particular village.2 1Department of Physics University of Minnesota Minneapolis. Grosberg1. and the major event was the all-Georgian wine testing festival. Levinthal paradox arises from the idea that the time required for a protein molecule to sample all of its conformations is at least Mτ. and τ is the time necessary to sample one conformation. The few participants (perhaps bad biophysicists) who were able to continue scientific observations realized soon that wine testing continued for an unexpectedly long time. Russia A long time ago. A. (2002) ©Adenine Press (2002) An Opinion Piece: Conversation on Levinthal Paradox & Protein Folding #3 A Few Disconnected Notes Related to Levinthal Paradox http://www. and assuming there were some M pavilions. the paradox goes. that cooperative (all-ornone) character of folding and unfolding transition is indicative of the sequences selected. In the most standard formulation. for instance. advertised as a merry traditional peasant holiday. Then. the month was October. are unlikely to realize the completion of the task and to stop at that. and their intent was to test systematically all different sorts of wine.edu 317 . unguided folding into one particular (native) state requires at least time of order Mτ which is far too long. This reinforces the understanding of Levinthal paradox which emerged in the last decade. Issue Number 3. This simple property of random walks and diffusion processes seems to be underappreciated in the current discussions revolving around the celebrated Levinthal paradox (1). simply because random walk visits some sites great many times before the first visit to some other sites. MN 55455. and each offering (for free!) a glass of young wine. Many biophysicists deem themselves experts. Berezovsky and E. well before the breakup of Soviet Union.
 This number. Levinthal did not say that the time of exhaustive conformation sampling (or wine testing) was Mτ . the very question is perfectly legitimate: what is the maximal length of the chain which can exhaustively sample all of its conformations within the specified time interval T. a sober person can do that. where s is a constant close to unity (see.318 Grosberg call it a “biological number. Leaving speculations aside for a moment. Then random walk of longevity t brings us as far as about √ t/τ. exhaustive sampling is only possible because the overall volume is restricted. reads t ≅ τM2 / 1nM. of course. Trifonov (2) turned the Levinthal’s argument up-side-down. Needless saying. This is the length between 9 and 15. Assuming exhaustive sampling time Mτ. let us discuss the estimate of N0 more closely. such as a millisecond to a second? Comparison with wine testing makes it immediately clear that the answer depends on how the sampling is organized. Clearly. In fact. and random walk is forced to come back. This increases the result for N0 to between 11 and 16.  twice smaller than (1).he said it was at least Mτ. which means there is no conformation dependent (free) energy landscape involved. This led IBET to a series of attractive speculations. When d crosses over 2. First of all. which seems to rule out most of the speculations by IBET. say. they wrote τesN0 = T and obtained N0 = (1/s) 1n (T/τ) . turns out to be somewhere between 23 and 31. Berezovsky and E. How can we estimate the exhaustive sampling time for the unbiased random walk model? Consider first that wine testing pavilions arranged along a line. Mτ is the time required to visit all pavilions in an orderly fashion. because random walk tends to leave behind large unvisited regions.” where biological numbers dwarf astronomical ones. e. never returning to the once visited conformation.g. one pavilion after another. For the random walk in the space of higher dimension d. in terms of wine testing. he said it is ≥ Mτ. according to (2). I. (Note that energy landscape. the mechanism of sampling changes.. To begin with. but sober model is unrealistic for the wine tester. which will not be derived here. which means we cover all M sites when √t/τ ≅ M. IBET would have obtained N0 = (1/2s) 1n (T/τ) . model of protein dynamics (and also of wine testing) would be purely random walk in the space of conformations. never returning to the already visited place. they decided to estimate the length of protein chain N0 such that at N < N0 protein can exhaustively sample all of its conformations within some specified time T. N: M = esN. This was sufficient for him to conclude that exhaustive sampling is impossible for realistic N. can be decreased). such as N = 150. the exhaustion time is larger than Mτ. Equally unrealistic is the model of protein chain dynamics which orderly samples all conformational states. it is definitely possible for sufficiently small N. and perhaps more realistic. At d > 2. Based on τM2. in other words. They argued that while exhaustive sampling of all conformations is out of reach for large N. . one millisecond. which. For all other sampling strategies. suppose it is an unbiased random walk. when present. the difference between τM and τM2 is very significant. and the time of exhaustive sampling is t ≅ τΜ2. Thus. In fact. accurate estimate of exhaustive sampling time by a random walk is not completely trivial. More sophisticated estimate for one dimensional case. M is so large because it is exponential in the degree of polymerization of the protein chain. will increase the exhaustion time (because exhaustion requires visiting all the tops of all energy barriers) – unlike folding time. Indeed. Of course. (4)). the result depends on d. The opposite.
There are quite a few other factors (5. Among random sequences.. in this case. because d = M corresponds to the situation where each conformation (site in conformation space. as already said. e. We leave it for the reader to decide whether the concept of folding should be applied to such a short chain. high cooperativity is a well established experimental fact (12).5. in the point of thermodynamic equilibrium between native and denatured states. It should be noted that the understanding of Levinthal paradox has progressed very significantly since it was first formulated (1). Indeed. Measurements in different regions of conformation space yield results for d between 1. Please do not forget that d here is the dimension of the abstract space of protein conformations. It is also well understood that high cooperativity is the property of proteins which is due to their peculiar selected sequences. is valid under the conditions of thermodynamic equilibrium. scales as τ exp (s´N2/3) (9-11). not the real three-dimensional space. which is very significantly smaller than Levinthal time proportional to τ exp (sN). In addition to this convincing argument by Finkelstein. and all fundamentally arising from the fact that we are dealing with a polymer chain in which all units are linearly connected. as it was first established by Shakhnovich and Gutin (13). while the parts – supposedly the units of hierarchical scenario – are not stable at all. His analysis seems quite convincing. Clearly. see also references therein).What is d in reality is anybody’s guess. it was foreseen by Bryngelson and Wolynes a long time ago (14). This latter fact has been extensively tested using lattice models (as described. in the review article (4). This estimate. or wine pavilion) can be equally probably reached from every other conformation in just one step τ. vast majority would not have exhibited any signs of cooperativity. His major point is that reasonably fast folding is frequently observed (see (8) and references therein) under the conditions where native state is not significantly lower in free energy (or not lower at all!) than fully denatured state – that is. Actually. this estimate is completely unrealistic. the all-or-none cooperative mechanism of folding. yielding N0 between 18 and 25. Speaking about the relation between sequence selection. even the globule as a whole is not particularly stable. At the next stage. exhaustion time would have scaled as τM ln M. 6). of all-or-none type. However. it is interesting to mention that experimental observations do not provide any evidence on the folding (under equilibrium conditions) time dependence on the chain length. This possibility was critically reviewed recently by Finkelstein (7). 319 Disconnected Notes Related to Levinthal Paradox . To conclude this part. under the conditions of thermodynamic equilibrium between folded and unfolded states.4 and 4.g. as the scaling of folding time. it appears that IBET significantly overestimated the length of a protein capable of exhaustive conformation sampling. all reducing the result for N0. it is found that the folding time. The relevant dimension was measured for the vicinity of the native conformation of a lattice model protein (5). That means. real protein chains are nowhere near this extreme. it is important to emphasize that the fact of non-cooperative folding in the majority of sequences is well understood beyond lattice models. Since everything related to the lattice models is perceived with a large dose of (healthy?) skepticism in protein community. It seems safe to say that this length is smaller than 15. First of all. Under such conditions. IBET suggested that exhaustively sampling blocks combine together to form hierarchically folding large proteins. the small value of N0 makes the stability of the presumed folding units questionable. which means that it relies on the transition between denatured and native forms being highly cooperative. The result close to (1) would be correct for the dimension d as high as M. and the whole of the hierarchical scheme even more difficult to imagine.
Fersht. Fraunfelder. Curr. I thank also V. USA 90. Pande.320 Grosberg the above mentioned theoretical prediction. Europhys. Sci. of proteins? There are very many works on these subjects. Their paper (2) initiated the present note. Levinthal. Letters 83. Bryngelson. 20. Struct. 12. at least) that the discussions about Levinthal paradox must now move forward to the new spheres. A. how precisely do these selected sequences slide down their folding funnels (17-20)? What are the physical principles behind the selection of certain spatial structures.. S. where every “good” sequence is capable of folding. 55. By contrast. Lett. 7195 (1993). Phys. Ptitsyn.. to make the list of them is a daunting task far beyond the framework of the present note. Grosberg. I. Proc. 594-600 (2001). Shakhnovich. A. it is getting increasingly clear that there are many sequences which meet the sufficient criteria of reliable folding. To summarize. Du. 7524-7528 (1987). τ exp (s´N2/3). V.. Ac. and do they have any relation to the principles involved in folding? What are the mechanisms of aggregation. Rev. Folding & Design 2. Proc. 7. Berezovsky I. B. J. How does the sequence selection work (or worked) in real evolution? What are the specific scenario of folding dynamics for selected sequences – how specific is the nucleation. The role of sequence selection is also well understood from a different view point. 4. it seems that the Levinthal’s question – how can protein sample “biologically large” number of conformations – has been answered: protein does not sample them. Adv. . Struct... Reviews of Modern Physics 72. Nunes Amaral.. Privalov. it seems clear (to the present author. 1989. 8. the selected sequences – the same ones which exhibit highly cooperative folding-unfolding transition! – are reliable in the sense that their native state with high probability survives and remains stable even after several mutations. E.. Grosberg... Berezovsky and E. A. Shakhnovich. 50-52 (1996). 3. 7. Most importantly. 6. Struct. A.. Chim. 7. T. Understanding this was a remarkable achievement of the last decade. S. Rev. Nat. 2. Finkelstein. 14. E. In the majority of sequences. J.. Sci. 1828-1831 (2000). A. Academic Press. T. Protein Physics. E. Gutin.. Biol. 17. Wolynes.. Acad. I. 34. 115 (1997). Scala. and Tanaka. Pande.. 10. J. 67 (1998). While the real mechanisms of evolutionary sequence selection remain unknown. 311-314 (2002). in the light of all the findings of the last decade. Yu.. 13. Biophys. 4670-4673 (1999). Tanaka. namely. R. Reference and Footnotes 1. Chem. A. what is the reaction coordinate associated with folding. Curr. A. Yu. 87. Opin. Finkelstein. Gutin. However many questions remain open. V. D. 44-45 (1968). V. Biol. Shakhnovich. 9. Proc. M. how many and which conformations belong to the transition state.. Yu. 2002. Opin. Protein. Trifonov E. 65. Sci. Natl. 12972 (1994). J. A. 33. P. Struct. Chem. Dyn. Folding & Design 3.... USA 91. Grosberg A.. 167-241 (1997). A. Pande for critical reading of the first draft of this manuscript. T. Shakhnovich. A. Dyn. G. 29-40 (1997). 16). Biomol.. I am indebted to I.. 5. and while the computational models of sequence selection keep improving since the first suggestions (15.. 5-6 (2002). 20.. 2002. P. in other words. or folds and fold families (21)? What are the general physical principles behind the enzymatic.. related to the mutation stability (see also in the review article (4)). there are sufficiently many “good” sequences for the evolution to select from. A. may be still an overestimate. 259-314 (2000). H. Biomol.. L. C.. V. M.. Biol. Colloquium talk at the University of Minnesota Physics Department. 11. R. T. 3-9 (1997). 16. Acad. Du. Grosberg. Phys. Badretdinov. October. 187. E. every mutation breaks the stability of the native state with the probability very close to 100%.. 15. Trifonov. Tanaka. Tanaka. Phys. Finkelstein.. O. I. A. L. Folding and Design 1. although sufficient to rule out any paradoxes. Letters 84. motor and other functions of proteins. Barthèlèmy. and my personal discussions with them were useful and pleasant. or mechanisms preventing aggregation. Nat. and does not need exhaustive conformation sampling to do so..
.. J. Wolynes. Luthey-Schulten. Folding and Design 1. K. S... Opin.. S. D... Biol. A. 68-79 (1998).. 2002 Communicated by the Editor Ramaswamy H Sarma .. Pande. N. Li. Struct. Chan. N. Z. 21. Tang. G. 441-450 (1996). 20. 321 Disconnected Notes Related to Levinthal Paradox Date Received: October 27.. Grosberg. H. Yu.. Onuchic.. Dill. 8. 19. D. C. S. T. Tanaka. Curr. H. Socci. Wingreen.18. Nat. Struct. R. Science 273. Helling.. V. 666 (1996). P. Biol. 4. A. 10-19 (1997). Rokhsar.. N.
Ames. and the assumption of a random coil as a starting point for the folding process is the basis of the Levinthal paradox. Struct. It suggests that the Levinthal paradox really does exist and has yet not been resolved. 5-6. the result of hydrophobic collapse. In our opinion the terminology “hierarchical folding” used by Finkelstein is improper. according to the most popular theories. Additionally what is important are not single loop contacts. The folding landscape does not resemble a flat golf course with a single hole corresponding to the native state. Finkelstein (9) does not support this simplest funnel-like mechanism of folding. folding nucleus Andrzej Kloczkowski Robert L. Issue Number 3. which greatly reduces the total conformational space (6-8). In the letter “Cunning simplicity of a hierarchical folding” Alexei V. Hierarchical folding usually refers to the old views on protein folding. but a highly interconnected network of such loop contacts.Journal of Biomolecular Structure & Dynamics. Berezovsky and Edward N. loop fold structure. which is untrue. ISSN 0739-1102 Volume 20. Trifonov (1) follows a long line of work. Biomol. Instead he explains the folding pathways through formation of a folding nucleus and a delicate thermodynamic balance between the native and denaturated states of the protein (10). 20. Dyn. looking for order or regularity in proteins. Rather it looks more like a bumpy funnel. closed loops. The common opinion in the protein folding community is that the Levinthal paradox (3) of finding a needle in a haystack doesn’t exist because proteins do not fold by randomly searching all possible (extremely large) numbers of conformations. being instead comparable to the size of the protein for single domain proteins.jbsdonline. which provides extra stability to a protein fold and which leads to their conservation in evolution. IA 50011-3020 The title “Loop Fold Structure of Proteins: Resolution of Levinthal paradox” of the communication by Igor N. with probably the most fundamental paper on this “paradox” written ten years ago by Zwanzig et al. evolutionary conservation. Contrary to Berezovsky and Trifonov (J. He also classified the idea of Berezovsky and Trifonov as a “hierarchical folding” and shows that such hierarchical folding would lead to a native state that is too stable to unfold. The funnel-like landscape of folding is. Levinthal paradox. Key words: protein folding. Proteins fold to the native state from non-native (denaturated) states that are already substantially structured and make all the arithmetic supporting the Levinthal paradox irrelevant. Jernigan* Baker Center for Bioinformatics and Biological Statistics Iowa State University 123 Office and Lab Bldg.com Abstract We show that loops of close contacts involving hydrophobic residues are important in protein folding. that the primary structure (sequence) leads to the formation of the protein secondary strucPhone: (515) 294-3833 Fax: (515)294-3841 Email: jernigan@iastate. A protein does not fold from random coil conformations. (2002) ©Adenine Press (2002) An Opinion Piece: Conversation on Levinthal Paradox & Protein Folding #4 Loop Folds in Proteins and Evolutionary Conservation of Folding Nuclei http://www. There is significant literature on this problem. 2002) the loops important in protein folding usually are much larger in size than 23-31 residues. (2). 5). so that the ball rolls almost always (except for kinetic traps) downhill (4.edu 323 .
We have developed a model for locating the evolutionarily conserved residues in proteins. Another possibility is that there are present three conserved subclusters. We studied the non-functional evolutionarily conserved residues in several subfamilies of proteins. The theory was also applied with success for identifying the conserved residues reported by Mirny and Shakhnovich (19) who used COC (conservatism of conservatism) method. What is however important are not the single loop contacts of regular size 23-31 . Using sperm whale myoglobin (1mbd) as the reference the structurally conserved residues are Val(10). As a matter of fact closed loops. respectively. Then we find the so called “supercore” inside the core. but not necessarily only those of almost regular size 23-31 as reported by Berezovsky et al. Such terminology leads him to conclude that the closed loops with nearly standard size segments of 23-31 residues reported by Trifonov and coworkers cannot be combined with more modern mechanisms of folding kinetics. The formation of such closed loops in the protein native state does not mean that residues forming such loop contacts always remain intact during the folding process. All these hydrophobic residues form a network of conserved contacts which is possibly the folding nucleus. Recently Kai-Li Ting and Jernigan (17) studied the conservation of the non-functional residues in the lysozyme/α-lactalbumin family and identified the possible folding nucleus. the conserved residues are Gly(6). pointing up the uncertainties involved in sequence comparisons. According to Ptitsyn this network of conserved contacts could be the folding nucleus for cytochrome c. These results show that looping contacts are indeed important for protein folding. Using the horse cytochrome c (1hrc) (which has the total length on 105 residues) as the reference for the numbering. The predicted conserved residues for 1hrc and 1mbd are exactly the same as those reported by Ptitsyn and Ptistyn and Ting. The non-functional conserved residues are located inside a protein core and their detection might give us valuable information about the mechanism of protein folding. such as a nucleation mechanism. in the series of their earlier papers (1113). We first find a core of a protein with the known structure based on the packing of residues. which in turn leads to the formation of the protein tertiary structure. 10-97 and 94-97. all of these conserved residues are obtained only by including phylogenetically diverse cases. Phe(10). They identified 19 conserved hydrophobic non-functional residues. Leu(94) and Tyr(97).324 Kloczkowski and Jernigan ture. Note that the sizes of these loops are mostly substantially larger than the 23-31 proposed by Berezovsky et al. but there is a possibility that these loop contacts inside a protein core might be a part of the folding nucleus (14). Ile(111). by maximizing the number of hydrophobic contacts. 10-94. Trp(14). which is of critical importance for its folding. The second subfamily of proteins studied by Ptitsyn and Kai-Li Ting was globins (16). He came to the conclusion that there is a common folding nucleus composed of four residues. especially if several of such loop contacts involving hydrophobic residues are located near one another inside the core. Kloczkowski and Jernigan (18) developed a theory of evolutionary conserved residues based on the hydrophobic-polar HP lattice model of protein and showed that a highly connected network of hydrophobic contacts provides extra stability to a protein fold and may be crucial in reaching the lowest energy native state. measured by number of contacts. That is probably too many due to a lack of substantial evolutionary diversity in the studied protein family. Leu(115). might be quite important in protein folding. All these residues are hydrophobic and form a network of five conserved contacts 6-94. This work was initiated in our Lab by the late Oleg Ptitsyn who studied different subfamilies of c-type cytochromes (15). Importantly. 6-97. Met(131) and possibly Leu(135). where each subcluster is composed of a network of conserved contacts.
146-150 (1995). Nauk SSSR 210. N. 1166-1180 (1999). 15. and Dill. M. E.. L. L. H. 20. 638-642 (1990). 177-196 (1999). L. 7524-7528 (1987). A. Proteins 23. Mol. I. J. Phys. 397-403 (2001).-L. 20-22 (1992). and Shakhnovich. Sci. 425-436 (2002). J. E. and Gutin. Berezovsky. 8... Finkelstein. K. 1419-1426 (2001). Zwanzig.. 307. O. C.. Proc. J. Ting. Berezovsky. A. A. O.. Sci. I... K. N.. A. N. 44-45 (1968). Badretdinov. J. I. Mol. Chim. I. Bahar. Acad. Finkelstein. Chim. Natl. 20. but possibly involving also short helical contacts.. A. Y. Natl. Proc. Ptitsyn. 19. 6.. and Trifonov. 5. 66. Chan. S. B. Natl. 7. USA 89. 17. 454-466 (1994). N. N. R. 325 Loop Folds in Proteins and Conservation of Folding Nuclei Date Received: November 6. Dokl. Proc. 54. Dyn. 9. reported by Berezovsky et al. K. Protein Sci.. 12. and Jernigan.. Ptitsyn. Biol.. Natl. 95. Lau. Mirny. A. USA 87. L. Dyn. J.. and Dill.residues. Acad. Struct. 291. Bryngelson. and Wolynes. 4. Struct. R. and Dill. Biol. J. K. Berezovsky. 8. J. A. G. K. and Bagchi. USA 84. A. Biomol. A. Kloczkowski. A. Yue. E.. 311-314 (2002). Sci. USA 92. E. Phys.-L. 3. N. J. V. In the case of 1hrc such a loop is of size 90 residues (which is nearly the total length 105 of the protein). 3775-3787 (1991). These are more consistent with the frequently remarked upon feature of proteins – that the ends of the chain are close together (20). Y. F. N. 14. 13. Levinthal. and Jernigan. 18. R. Acad. B. Dill. 11. I. 283-286 (2000). Biol. K.. Mol. R. J. B. Biomol. 291. Akad. 142-150 (1995). and Trifonov. 2002 Communicated by the Editor Ramaswamy H Sarma . J. N. Biomol.. Struct. 10. 278. A. 19. References and Footnotes 1. Sci.. Biophys. and Ting. J. D. Ptitsyn. also approaching the total length 153 of the protein.. but rather an interconnected network (or cluster) of such contacts of loops of much larger size. Evolutionary Conserved Residues and Protein Folding. P. Berezovsky. 655-666 (1998). 65. Biol. E. Mol. FESB Letters 466. Acad. Szabo. K. 20. Proc. and Trifonov. 671-682 (1999). and Trifonov. Biol. and Jernigan. Dyn. I. J. while in the case of 1mbd the size of the loops is 100-120. 16. Evol. V. to be published... 5-6 (2002). B. Mol.. K. O. Grosberg A. Chem. 1213-1215 (1973). 2..
ISSN 0739-1102 Volume 20. In particular. Another point of discussion concerned the question of whether protein folding is under thermodynamic or kinetic control. Indeed.com Abstract We would be tempted to state that there has never been a Levinthal paradox. (2002) ©Adenine Press (2002) An Opinion Piece: Conversation on Levinthal Paradox & Protein Folding #5 What is Paradoxical about Levinthal Paradox? http://www. Anfinsen did not restrict nucleation to local interactions along the protein chain. He did not seem to find this paradoxical and immediately proposed a straightforward solution. (5) in 1976. Anfinsen clarified this point in 1973 by introducing the concept of ‘flickering equilibria’ (2): “it seems reasonable to suggest that portions of a protein chain that serve as nucleation sites for folding will be those that can ‘flicker’ in and out of the conformation that they occupy in the final protein. It was realized at about the same time that the folding of a protein into its native structure essentially occurs independent of the initial conditions. 50 avenue Roosevelt B-1050 Bruxelles. This ruled out the hypothesis. Levinthal solved his own paradox. “proteins fold by following a multiply branched pathway”.” Moreover. this suggests local amino acid sequences which form stable interactions and serve as nucleation points in the folding process. pleated sheets or β-bends”. to our knowledge originally due to Chantrenne (4). In other words. as he realized that proteins have no time to explore exhaustively their conformational space on the way to their native structure. Levinthal himself solved what could at that time appear as nonintuitive. Clearly.” Of course.ac. In the 70’s.jbsdonline. The number of pathways was (and is still) a subject of debate. there are two words in Levinthal’s sentence that are questionable: local and stable. that the only way of achieving correct folding is by the sequential growth of the polypeptide chain on the ribosome.Journal of Biomolecular Structure & Dynamics. Belgium During a meeting held in 1969 in Monticello. which has essentially never been refuted. Let us start with the last one. Marianne Rooman* Yves Dehouck Jean Marc Kwasigroch Christophe Biot Dimitri Gilis Ingénierie Biomoléculaire Université Libre de Bruxelles CP 165/64. Issue Number 3. he proposed that “protein folding is speeded and guided by the rapid formation of local interactions which then determine the further folding of the peptide. In the same report (usually incorrectly referenced). according to Honig et al. this conclusion was reached on the basis of the experimental observation that small denatured proteins were able to refold in vitro (3).be 327 . most current scenarios involve a huge number of parallel pathways possibly sharing a number of key steps. Levinthal raised an interesting problem about protein folding. since he stated that “the nucleation centers might be expected to involve substructures as helices. According to Anfinsen (2). In good agreement with this assumption. Levinthal estimated the number of different conformations accessible to a 150-residue protein to be roughly of the order of 10300. a few residues can only present a very marginal stability. even paradoxical. whereas the number of conformations sampled by a natural protein before reaching its final state is of the order of 108 (1). under suitable conditions. and that they will form a relatively rigid structure stabilized by a set of cooperative interactions. native protein *Phone: 32-2-650 2067/5572 Fax: 32-2-650 3606 Email: mrooman@ulb. the prevailing view was that folding follows pathways along which nucleation events take place. Indeed.
In the context of hierarchic folding. there has been a continuing dispute between the supporters of nucleation and those of hierarchic folding. numerous experimental and theoretical studies have revealed protein sequences exhibiting a strong signal towards the native structure. and indisputably provide valuable precisions and clarifications on the mechanisms of protein folding. which are much less numerous than high energy states. much of the controversy vanishes. Another much debated issue is whether nucleation centers consist of local interactions along the chain or of tertiary contacts. these approaches should be considered as complementary. rather. Another concept proposed a few years ago is that protein energy landscapes have the shape of a funnel (11). What have we learned since then? Many new folding models. This unquestionably yields a very nice. a necessary condition. We feel however that the originality of these ‘new’ concepts is often somewhat overrated by comparing to a hypothetical ‘classical view’ of protein folding which would involve well defined pathways. with for example small flickering secondary structure elements forming a tertiary contact and thereby inducing nucleation. which have been assembled during evolution. in specific tertiary contacts. theories and concepts have been proposed.” These chosen extracts show that the folding problem was well understood in the early 70’s. as nicely pointed out by Anfinsen (2). Therefore. in general. This is supported by the experimental observation that some peptides are more structured in solution than others (7). with this softened view. Curiously however. This is probably. some specific protein residues have been observed to form native tertiary contacts earlier than others and to stabilize folding nuclei (8). encoded locally along the sequence or. the idea that original proteins were small closed-loop peptides with flickering stability. vision of the folding mechanism. Moreover. it has been suggested that the energy gap between the native conformation and the other conformations that are structurally unrelated to it must be sufficiently large (10). . To achieve rapid folding towards the native state. However. Indeed. debates around Levinthal’s paradox. predominance of local interactions and an absolute necessity for stable intermediates. which actually means that they flicker in and out of a specific conformation. intuitive. depending or not on the environmental conditions. The answer seems obvious: it is proteindependent. First. often based on interesting ideas or experimental observations. is quite attractive. where folding is funneled towards low energy states. Again. As stressed by Honig (6). on the contrary.328 Rooman et al. It is difficult to believe that all current proteins exhibit this property. it is obvious that all residues along the chain are not equally prone to constitute nucleation centers. and that some trace of this evolution is left in the current proteins. Both tendencies are probably often conjugated. we do not feel that hierarchic folding must be opposed to nucleation. structures correspond to global free energy minima. frequently opposing the partisans of different models. But Levinthal (1) argued that “the final conformation has not necessarily to be the one of lowest free energy. where typically some secondary structure elements or loops form first. hierarchic folding units must not be viewed as rigid but rather as flickering entities. It obviously must be a metastable state which is in a sufficiently deep energy well to survive possible perturbations in a biological system. this simplistic view is very unlikely to correctly reflect the thoughts prevailing in those pioneering days. the lack of contradiction between old and new views has not prevented ongoing. Hence. These two views easily reconcile when considering that small structure elements can only flicker in and out. On the other hand. passionate. at least in a slightly modified form taking into account the existence of proteins adopting several folded structures. it has been recently suggested that proteins are made up of closed loops that fold separately (9).
Levinthal. P. E. 9. R.. Natl. 14. Dyn. N. exhibit domain swapping or several folded structures. Shakhnovich.. E. Biol.. & Munck E. These considerations bring us to think about the folding mechanism that evolution tends to favor. Natl. Acad. Gutin. p 122. M. P. A.. 235. and Trifonov. London. H. Wilson. This issue is probably related to the possible biological role played by the multiplicity of conformations. The development of potent experimental and theoretical techniques have led to support and clarify many of the proposed views. We have. Biomol. A. A. E.. J... 1961). J. Abkevich.. FEBS Lett. Bryngelson. J.. Honig.. with the finding of proteins that polymerize. A. C.. V. recent advances should somewhat be relativized.. 223-230 (1973). E. 20. and Karplus. B. Proteins 21. having adequate folding and functional properties. N. H.. Tsibris J. J. and Levinthal. Proc. Acad. J. E. I. A. and Anfinsen. Biol. R. or to its pathological consequences. Biol. 2. 886-887 (1970). C... A. (Academic Press. Nature 318. E. 11.. Biol. 4. and M. J. Mol.. A. and Shakhnovich.. 3.. pp 153-166. Oxford.. V. Natl.. 283-293 (1999). Proceedings of a meeting held at Allerton House. Edited by Debrunner P. USA 47. pp 22-24.. Science 167. I. are Research Assistant and Research Director. B. Mol.. R. Bogatyreva. and that there is probably also not a unique answer to the question whether native structures correspond to relative or absolute free energy minima. A. N. and to translating the ‘old view’ in the framework of statistical mechanics. the impression that some earlier contributions in the disciplne have been forgotten or misinterpreted and that in light of these. D.However.. Wright. Urbana. Chen.. aggregate. F. M. 7167-7175 (1988). 283-286 (2000). Grosberg. D. Cross.. N. Wright. C. A. R. Chantrenne H. J. Sci. 201. (University of Illinois Press. Mol. J. 466. 521-523 (2001).. 201-217 (1988). Besides. evolution singled out a tiny fraction of possible amino acid sequences. J. 167-195 (1995). I.. and Finkelstein. Houghten. edited by Vogel. New-York & Paris. Sali. 1282-1286 (1995). Biochemistry 27. Mol. S. 7. C. Socci. References and Footnotes 1. J. 12. J. 1614-1636 (1994).. 8.. D. however. Ray. 2002 Communicated by the Editor Ramaswamy H Sarma . A. 1969). respectively.. Acad. 260-288 (1995). 329 What is Paradoxical about Levinthal Paradox? Date Received: October 29. B.. Houghten R.. Itzaki.. Rance. Mol. B. N. P. M. by a grant from the Fonds de la Recherche pour l’Industrie et l’Agriculture (FRIA). How to Fold Graciously. General remarks on protein structure and biosynthesis. a funnel-like shape has been shown to constitute an insufficient condition for ensuring consistent folding (12). Acknowledgments D. In Mossbauer spectroscopy in biological systems. 254. Illinois. Anfinsen. Dyson. 6.. Science 181.. 1963). H. R. and Fersht. it was realized that folding is even more complex to predict and simulate than originally hoped for. 1309-1314 (1961). L. Onuchic. N. B. N. this basically corresponds to requiring the independence towards initial conditions and the absence of insurmountable energy minima on the ways to the native state. H. 5. and Lerner.. A.. 10. Pretending that nothing has evolved since the early 70’s is certainly an exaggeration. Y. Bryson. M. The Biosynthesis of Proteins (Pergamon Press. 480-483 (1985). Schechter. Berezovsky. Anfinsen. Wright. and Lampen. Struct. Monticello. and White F. R.. Illinois.. H. Haber. Protein Eng. A. Otzen D. J. G.. In Informational Macromolecules. NewYork & London... Biol. USA 73. But most certainly.. and Y. A. 307. J. V. I. Trifonov. G. O. Finally. Proc. E. is supported by a BioVal research program of the Walloon Region. 5-6 (2002). C. and Lerner. I. and Wolynes P. Proc. Dyson. at the Belgian National Fund for Scientific Research (FNRS). K. R. Honig B. Sci. S. E.. J. B. C. 1419-1426 (2001).. 1974-1978 (1976).. Anfinsen. 293. E. N.. Berezovsky. USA 92. and Lerner. Sela. Dyson. C.
that the sum of infinite rational numbers might be finite. those producing an over-all structure at odds with excluded volume). Levinthal argued that since the number of possible conformations of a protein chain may be estimated to be exponential in the number (N) of aminoacids.e. i. nonhierarchical. NY 10027 3100 Morningside Drive New York. we believe. Since the layman would find the latter statement more striking than the original premise (we assume he has not been exposed to Calculus). and Economics Program Columbia University Business School 3022 Broadway #8G New York. NY 10027 *Phone: 773 834 4782 Fax: 773 702 0439 Email: ariel@uchicago. we decided to coin one of our own: A paradox is a logically consistent construction which sprouts from a false premise but one which is not too obviously so. Thus. after this thinking induced by the paradox. paradoxes help us to think more rigorously and be more critical of our own thoughts. the repulsive LennardJones terms in the intramolecular potential energy – to name a single contribution – dramatically reduce the probability of certain conformations (i.com Abstract In this contribution we shall try to argue that no folding scenario – be it hierachical. And. he might be a bit less of a layman. causing surprise and forcing us to revise the starting premise. In this way. he might be surprised at some of Zeno’s conclusions.Journal of Biomolecular Structure & Dynamics. The probability of a conformation of an individual aminoacid within the chain depends on the geometric or structural constraints and basins of attraction the chain generates as each aminoacid picks its coordinates. Thus. (2002) ©Adenine Press (2002) An Opinion Piece: Conversation on Levinthal Paradox & Protein Folding #6 Protein Folding: Where is the Paradox? http://www. Here is. where the Socratic or moral content of the paradox resides. like “an arrow never reaches the target”. Not quite in the ancient tradition of Zeno’s paradoxes. nucleation. and that the conformations available to each individual aminoacid may be assigned constant and equal probabilities (although the equality condition may be relaxed) at all times throughout the exploration of conformation space. nor are their probabilities constant in time. while the attractive pairwise contributions dramatically increase the probability of other conformations. and produces a logical conclusion which is very ostensibly false. etc. He assumed the number of possible conformations for each individual aminoacid to be small and fixed. Thus. say from 2 to 100. IL 60637 2Finance Since we could not find a satisfactory definition of paradox. This premise is false: the conformations of an individual aminoacid are not equally probable in time.* Alejandro Belinky2 María de las Mercedes Boland3 1Institute for Biophysical Dynamics The University of Chicago Chicago. he will be forced to think carefully about something he might have never thought about otherwise. ISSN 0739-1102 Volume 20. and for that reason. the exhaustive exploration of conformation space in a finite time of biological relevance is practically impossible since it is also exponential in N. the layman might not know that the sum of infinite rational numbers may yield a finite result. – needs to be invoked to solve Levinthal’s paradox: It fails on its own grounds.e.jbsdonline. Issue Number 3. but this does not imply that the time to exhaustively visit all accessible conformations of the chain is also exponential in N. the number of a-priori possible conformations of the chain might indeed be exponential in N. Ariel Fernández1.edu 331 .
2002 Communicated by the Editor Ramaswamy H Sarma . even the idea that the protein chain exhaustively explores all conformations available along a successful folding pathway is false. Date Received: October 5. Thus. Why is the thermodynamic limit even relevant to protein folding? Where does the idea that the native structure is the free energy minimum come from? To the best of our knowledge. The former might not be much of a defect in a paradox.332 Fernández et al. we fail to see why the Levinthal paradox attracts attention: Its premises are too obviously false and the striking conclusion it purports to reach is uncalled for. these are baseless hypotheses. In a nutshell: The very existence of an intramolecular potential renders the starting premise of the Levinthal paradox false. the latter certainly is. Moreover.
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