Folding of circular permutants with decreased contact order: general trend balanced by protein stability 1 1Edited by A. R. Fersht

Lindberg, Magnus; Tångrot, Jeanette; Otzen, Daniel E.; Долгих, Д. А.; Finkelstein, Alan; Oliveberg, Mikael

doi:10.1006/jmbi.2001.5186

Cited by 56 publications

(57 citation statements)

References 37 publications

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“…One possible explanation for our results is that the destabilization of the permutants results in a deceleration of the refolding process that opposes the acceleration of refolding caused by the decrease in RCO. Lindberg et al (15) examined two circular permutants of S6, the folding rates of which in water were similar also, varying by a factor of 1.7. They analyzed their data by comparing folding rates at the denaturant midpoints rather than under the same conditions; when adjusted for stability, the folding rates do vary and are in closer agreement with the expectations from the RCO correlation.…”

Section: Discussionmentioning

confidence: 99%

“…However, a similar comparison of the folding rates of the naturally occurring examples of two-state proteins at the denaturant midpoints results in a weaker correlation with RCO than comparing the rates extrapolated to water (R ϭ 0.79 vs. 0.89 from table 2 of ref. 15). The poorer correlation at iso-energetic conditions is consistent with the observation that protein stability does not correlate with folding rates among the set of two-state proteins.…”

Section: Discussionmentioning

confidence: 99%

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Experimental evaluation of topological parameters determining protein-folding rates

Miller

Fischer²,

Marqusee³

2002

Proc. Natl. Acad. Sci. U.S.A.

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Recent work suggests that structural topology plays a key role in determining protein-folding rates and pathways. The refolding rates of small proteins that fold without intermediates are found to correlate with simple structural parameters such as relative contact order, long-range order, or the fraction of short-range contacts. To test and evaluate the role of structural topology experimentally, a set of circular permutants of the ribosomal protein S6 from Thermus thermophilus was analyzed. Despite a wide range of relative contact order, the permuted proteins all fold with similar rates. These results suggest that alternative topological parameters may better describe the role of topology in proteinfolding rates.T he amino acid sequence of a protein and its chemical environment determine its native structure (1), but how this structure is determined from sequence remains one of the major unsolved problems in biology. The process of protein folding cannot be accomplished by random search through all possible conformations, because the number of structures available to an unfolded protein is too large; therefore there must be a biased or directional search in order for a protein to reach its native state. Small Two-State Proteins Show a Wide Range in Folding RatesThe simplest models for studying the protein-folding process are those that refold without intermediates (2). To date there are more than 20 examples of such simple systems. These proteins all fold cooperatively in a mono-exponential fashion from the denatured state, but the rates at which they fold span 6 orders of magnitude. What causes this remarkable variety of refolding rates? Folding Algorithms Based on the Topology of the Native State Have Predictive ValueRecently, several folding algorithms based on native structural information have been used to predict folding rates and nucleation sites (3-6). These efforts suggest that the topology of the final structure is an important determinant in the mechanism of protein folding.If native topology plays a major role in protein folding, is there a simple structural parameter that will capture this feature and explain the large range of observed folding rates? Recently, several simple parameters defining topological features of the native state have been shown to correlate well with proteinrefolding rates. The first and most commonly used parameter is relative contact order (RCO; ref. 7). Remarkably, RCO, which represents the normalized average sequence separation between contacting residues, was observed to correlate extremely well with the folding rates of a small set of two-state proteins. The lower the RCO, the faster the protein folds. Although it is possible to alter folding rates significantly through point mutations that do not change RCO, and there are proteins with similar RCOs and different folding rates (8), in general this correlation has improved as more two-state proteins have been characterized (9) and can explain the difference in folding rates among a family of structurally homologous pro...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Experimental evaluation of topological parameters determining protein-folding rates

Miller

Fischer²,

Marqusee³

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…To facilitate the comparison between the different constructs the wild-type numeration was used for all point mutations in Results and Discussion ( Table 2). The permutants P [13][14] and P 68-69 were designed and constructed as described (43). The third permutant, P 54-55 , was constructed from P [13][14] by making an incision between residues K54 and D55 in the loop connecting the ␤2 and ␤3 strands, adding a methionine in the new N terminal and restoring the wild-type ␤1-␣1 loop.…”

Section: Methodsmentioning

confidence: 99%

Identification of the minimal protein-folding nucleus through loop-entropy perturbations

Lindberg

Haglund

Hubner

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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To explore the plasticity and structural constraints of the proteinfolding nucleus we have constructed through circular permutation four topological variants of the ribosomal protein S6. In effect, these topological variants represent entropy mutants with maintained spatial contacts. The proteins were characterized at two complementary levels of detail: by -value analysis estimating the extent of contact formation in the transition-state ensemble and by Hammond analysis measuring the site-specific growth of the folding nucleus. The results show that, although the loop-entropy alterations markedly influence the appearance and structural location of the folding nucleus, it retains a common motif of one helix docking against two strands. This nucleation motif is built around a shared subset of side chains in the center of the hydrophobic core but extends in different directions of the S6 structure following the permutant-specific differences in local loop entropies. The adjustment of the critical folding nucleus to alterations in loop entropies is reflected by a direct correlation between the -value change and the accompanying change in local sequence separation.O ur current understanding of the protein-folding nucleus is based on a synthesis of results from simulation (1) and experimental mapping of site-specific contacts in the transitionstate ensemble by protein engineering (2, 3). From these results it is apparent that the free-energy landscape controlling the folding process is highly evolved, with few traps and a characteristic bias toward native contacts (4, 5). Consistent with the general insensitivity of the transition-state structure to point mutation and the remarkable success of reproducing experimental data with simplistic Go models, the prediction from such biased landscapes is that the sequence of folding events is largely determined by the topology of the native structure (1, 4, 6, 7). One intriguing possibility is that folding follows a trajectory of the lowest successive loop-entropy cost (8, 9). To directly test this idea we have analyzed the folding behavior of four S6 variants in which the loop-entropy cost of forming pairwise contacts has been systematically altered through circular permutation (10-12). The results show that the -value distribution defining the S6 nucleus is plastic and responds to circular permutation in a systematic manner. Contacts are recruited in directions with decreased sequence separation, and contacts are lost at the entropically penalized regions of the backbone incisions. Even so, the critical nuclei of the permuted proteins share a minimal two-strand-helix motif with variable but overlapping composition of secondary-structure elements. Moreover, it is apparent that a specific number of side-chain contacts are required to turn the folding free energy profile downhill, and that the dimension of this cluster matches the size of the smallest cooperatively folding proteins. Results and DiscussionPronounced Changes of the -Value Distribution upon Circular Permutation. Ch...

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“…Folding studies on artificially permuted proteins have been specifically aimed at monitoring the effect on both folding speed and mechanism. By systematically altering the sequence connectivity of the ribosomal protein S6, Oliveberg and coworkers (12) showed that protein folding rate constants of circularly permuted variants are well predicted by the contact order parameter. On the other hand, analysis of protein folding pathways reveals apparently contradicting results.…”

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confidence: 99%

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

Ivarsson¹,

Travaglini-Allocatelli

Brunori

et al. 2008

Journal of Biological Chemistry

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One of the most extreme and fascinating examples of naturally occurring mutagenesis is represented by circular permutation. Circular permutations involve the linking of two chain ends and cleavage at another site. Here we report the first description of the folding mechanism of a naturally occurring circularly permuted protein, a PDZ domain from the green alga Scenedesmus obliquus. Data reveal that the folding of the permuted protein is characterized by the presence of a low energy off-pathway kinetic trap. This finding contrasts with what was previously observed for canonical PDZ domains that, although displaying a similar primary structure when structurally re-aligned, fold via an on-pathway productive intermediate. Although circular permutation of PDZ domains may be necessary for a correct orientation of their functional sites in multidomain protein scaffolds, such structural rearrangement may compromise their folding pathway. This study provides a straightforward example of the divergent demands of folding and function.A crucial development of our knowledge on protein folding has been contributed by correlating rate constants of folding of small proteins with their topology as measured by the gross parameter of the contact order (1). The contact order represents the average distance, on the primary structure, between interacting residues in the tertiary structure. A protein with a low contact order will by and large present interacting residues that are close in sequence. On the other hand, high contact order implies a large number of long-range interactions. Baker and coworkers (2) first showed a strong correlation between kinetic and structural parameters, suggesting that protein topology is a key factor in determining the folding pathways and speed. An important corollary of these observations is that folding transition states must reflect a distorted version of the native state. This feature was already captured by the nucleation condensation model (3, 4), which suggested the protein to fold all at once around an extended, weakly formed, folding nucleus.The notion that protein folding pathways are governed by protein topology (1) has recently been challenged by ingenious experiments using topological mutants such as circularly permuted variants (5-9). Despite the dramatic change experienced by the primary structure, circular permutations seem well tolerated by several protein sequences. In nature, circular permutations have been recognized in ϳ5% of proteins of known structures (10,11). Folding studies on artificially permuted proteins have been specifically aimed at monitoring the effect on both folding speed and mechanism. By systematically altering the sequence connectivity of the ribosomal protein S6, Oliveberg and coworkers (12) showed that protein folding rate constants of circularly permuted variants are well predicted by the contact order parameter. On the other hand, analysis of protein folding pathways reveals apparently contradicting results. In particular, while the folding pathway of chymotr...

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Folding of circular permutants with decreased contact order: general trend balanced by protein stability 1 1Edited by A. R. Fersht

Cited by 56 publications

References 37 publications

Experimental evaluation of topological parameters determining protein-folding rates

Experimental evaluation of topological parameters determining protein-folding rates

Identification of the minimal protein-folding nucleus through loop-entropy perturbations

Folding and Misfolding in a Naturally Occurring Circularly Permuted PDZ Domain

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