Different folding transition states may result in the same native structure

Viguera, Ana Rosa; Serrano, Luís; Wilmanns, Matthias

doi:10.1038/nsb1096-874

Cited by 215 publications

(228 citation statements)

References 29 publications

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“…Moreover, the folding mechanisms of globular proteins are generally robust; only a drastic change in the energetic balance, such as by circular permutation, can in some cases shift the folding nucleus from one part of the structure to another (27)(28)(29)(30). By contrast, the experimental results for myotrophin presented here and for the 7-ANK protein Gankyrin (R. D. Hutton, A.R.L., and L.S.I., unpublished results) and the 12-ANK protein D34 (N. D. Werbeck and L.S.I., unpublished results) reveal parallel folding routes of similar energy, and simulations carried out on a number of ANK proteins also predict pathway heterogeneity (26).…”

Section: Discussionmentioning

confidence: 99%

Rational redesign of the folding pathway of a modular protein

Lowe

Itzhaki

2007

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

100

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The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats. However, close examination of the mutant chevron plots reveals that simple models are insufficient to describe all of the data, and double mutant analysis subsequently confirms that there are parallel folding pathways. Destabilizing mutations in the C-terminal repeats reduce flux through the wild-type pathway, making a new route accessible in which folding is initiated at the N-terminal repeats. Thus, the folding mechanism of the repeat protein is poised on a fulcrum: When one end of the molecule is perturbed, the balance shifts between the different nucleation sites. The vast majority of studies on small globular proteins indicate a single, well defined route between the denatured and native states. By contrast, the potential to initiate folding at more than one site may be a general feature of repeat proteins arising from the symmetry inherent in their structures. We show that this simple architecture makes it straightforward to direct the folding pathway of a repeat protein by design.ankyrin repeat ͉ myotrophin ͉ ⌽ value ͉ protein engineering ͉ parallel pathways R epeat proteins, such as ankyrin repeats, leucine-rich repeats, and tetratricopeptide repeats, consist of tandem arrays of a structural motif of 20-40 aa that stack in a roughly linear fashion, creating elongated and superhelical architectures (1-3). Repeat protein structures are composed entirely of short-range interactions, either within a repeat or in adjacent repeats, in striking contrast to the more complex topologies of globular proteins that are stabilized by contacts between residues distant in sequence (4). They are ubiquitous, representing Ͼ5% of eukaryotic proteins in the Swiss-Prot database, and highly versatile, mediating molecular recognition in many different biological processes, but little is known about their folding mechanisms. The simple, modular nature of these structures has made them uniquely successful as scaffolds for engineering novel binding specificities (5-7). Does the repeat architecture likewise give rise to distinct folding properties, and are their folding pathways also amenable to design?In this study, we have addressed this question using the small, well behaved ankyrin (ANK) repeat protein myotrophin. The folding and unfolding properties of ANK repeats are of particular interest because these motifs are thought to play a role in mechanical signal transduction by virtue of their putative elastic properties (8, 9). The ANK repeat is a 33-residue motif consisting of a pair of antiparallel ␣-helices linked to the neighboring repeat via an antiparallel ␤-loop. The 11...

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Section: Discussionmentioning

confidence: 99%

Rational redesign of the folding pathway of a modular protein

Lowe

Itzhaki

2007

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

100

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“…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)(11)(12). The results show that the -value distribution defining the S6 nucleus is plastic and responds to circular permutation in a systematic manner.…”

Section: Circular Permutation ͉ -Values ͉ Transition Statementioning

confidence: 99%

“…3 and Table 4, which is published as supporting information on the PNAS web site). Evidence that the coupling between -values and loop entropy is universal is provided by an earlier analysis of circular permutants of the ␣-spectrin Src homology 3 domain (10,41) where the application of Eq. 3 to the literature data yields R ϭ 0.76 (Table 4 and Table 5, which is published as supporting information on the PNAS web site).…”

Section: Malleable Contact Recruitment Indicates Broad Folding Progrementioning

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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“…It may be thought of as joining the ends of a protein and then opening the chain elsewhere. These experiments have shown that it is possible in some cases to permute the termini of a protein and still allow folding into the native conformation (1,2), thus providing valuable information on protein folding. In practice the conceptual "circular proteins" intrinsic to the design of circular permutants are never actually synthesized but are theoretical intermediates.…”

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confidence: 99%

Acyclic Permutants of Naturally Occurring Cyclic Proteins

Daly

Craik

2000

Journal of Biological Chemistry

104

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Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple-stranded ␤-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the ␤-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

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Different folding transition states may result in the same native structure

Cited by 215 publications

References 29 publications

Rational redesign of the folding pathway of a modular protein

Rational redesign of the folding pathway of a modular protein

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

Acyclic Permutants of Naturally Occurring Cyclic Proteins

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