Are proteins made from a limited parts list?

154

304

There is growing appreciation of the functional relevance of unfolded proteins in biology. However, unfolded states of proteins have proven inaccessible to the usual techniques for high-resolution structural and energetic characterization. Unfolded states are still generally conceived of as statistical coils, based on the pioneering work of Flory [(1969 T he process by which a protein acquires its native structure is among the most complex reactions known, and challenges remain in defining the nature of the transition state(s), the structure and role of intermediates, and the properties of the starting ensemble of states (1-4). According to Flory (5) and Tanford (6), unfolded proteins can be represented as statistical random coils, in which a given residue has no strong preference for any specific conformation. Confirming earlier conclusions by Tiffany and Krimm (7-9), recent evidence from a variety of spectroscopic probes (10-22), theoretical studies (23-34), and coil library surveys (35-43) consistently point to a major role for the polyproline II (PPII, ⌽ ϭ Ϫ75°, ⌿ ϭ ϩ145°) conformation in oligo-Ala (for review, see ref. 3 and related articles in the same volume), oligo-Lys, and oligo-Glu peptides (44). We have reported that in a seven-Ala peptide model PPII converts to a ␤-like structure with increasing temperature (13). These findings raise several important questions regarding the structure of unfolded proteins: Although alanine is arguably a reasonable model for the unperturbed peptide backbone, is PPII also present in unfolded peptide chains composed of nonalanine nonproline residues? Is there an intrinsic PPII propensity for each individual side chain? If PPII is in equilibrium with ␤-structure, is there a correlation between scales of PPII propensity and analogous ␤-sheet scales? To what extent is PPII sequence and context dependent?Here, we address these questions by analyzing a series of end-blocked host pentapeptides AcGGXGGNH 2 , where X denotes 19 natural amino acids except glycine. Members of the series are found to differ in their extent of PPII conformation as determined by NMR and CD spectroscopy. Our results lead to the following conclusions: PPII is present as a dominant conformation in the majority of AcGGXGGNH 2 peptides. Different side chains show distinct propensities to adopt PPII in these unfolded molecules. Importantly, we find an inverse correlation between the determined PPII scale and the ␤-sheet-forming propensities derived from a zinc-finger model system (45) when 18 aa (except Gly and Pro) are divided into two groups: one, the nonpolar ␤-branched and bulky aromatic residues (VIWFY) and the other all of the remaining side chains. Finally, we find a correlation between our PPII scale in AcGGXGGNH 2 and a PPII scale derived from alternative model peptides such as AcPPPXPPPGYNH 2 (46). Still there are indications that the PPII scale is likely to be sequence and context dependent (47). Materials and MethodsPeptides Synthesis and Purification. Peptides were assembled on Rink Amide res...

Section: Ppii Contents and The Correlation Of Ppii Propensities With supporting

confidence: 52%

Polyproline II propensities from GGXGG peptides reveal an anticorrelation with β-sheet scales

Shi

Kang

Liu

et al. 2005

154

304

“…Our results suggest that there are cases for which we must extend this notion of a random coil with residual structure even to the collapsed unfolded state, populated under conditions that have so far evaded confrontation with the ''reconciliation problem'' (6). Compaction of the chain would be expected to contribute to the formation of local structure, because the increase in excluded volume effects will introduce more steric interference with nonnearest-neighbor residues (29,60). As a result, the backbone will be forced even more into the core regions of the Ramachandran map, corresponding to extended structures that avoid such steric conflicts.…”

Section: Discussionmentioning

confidence: 84%

“…Clearly, global random coil behavior does not exclude the presence of short structured segments (30,(56)(57)(58), even more so if these are only populated transiently. This argument has been used to resolve the seemingly conflicting views of residual structure observed in proteins under highly denaturing conditions on the one hand and the successful description of global properties of unfolded polypeptides with the random coil model on the other (59,60). Our results suggest that there are cases for which we must extend this notion of a random coil with residual structure even to the collapsed unfolded state, populated under conditions that have so far evaded confrontation with the ''reconciliation problem'' (6).…”

Section: Discussionmentioning

confidence: 99%

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Hoffmann

Kane

Nettels

et al. 2007

217

338

We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...

“…In any case, the number of conceivable paths for even a small protein of 100 residues is of order at least 10 30 and possibly much larger (18). At every time slice, each molecule in the population will have a specific conformation, but with only small energy barriers between them (approximately kT; k is the Boltzmann constant; T is the absolute temperature), so conformations are readily interconvertible.…”

Section: Part 1 Protein Folding: the Current Perspectivementioning

confidence: 99%

A backbone-based theory of protein folding

Rose

Fleming

Banavar

et al. 2006

Self Cite

457

412

Under physiological conditions, a protein undergoes a spontaneous disorder º order transition called "folding." The protein polymer is highly flexible when unfolded but adopts its unique native, three-dimensional structure when folded. Current experimental knowledge comes primarily from thermodynamic measurements in solution or the structures of individual molecules, elucidated by either x-ray crystallography or NMR spectroscopy. From the former, we know the enthalpy, entropy, and free energy differences between the folded and unfolded forms of hundreds of proteins under a variety of solvent͞cosolvent conditions. From the latter, we know the structures of Ϸ35,000 proteins, which are built on scaffolds of hydrogen-bonded structural elements, ␣-helix and ␤-sheet. Anfinsen showed that the amino acid sequence alone is sufficient to determine a protein's structure, but the molecular mechanism responsible for self-assembly remains an open question, probably the most fundamental open question in biochemistry. This perspective is a hybrid: partly review, partly proposal. First, we summarize key ideas regarding protein folding developed over the past half-century and culminating in the current mindset. In this view, the energetics of side-chain interactions dominate the folding process, driving the chain to self-organize under folding conditions. Next, having taken stock, we propose an alternative model that inverts the prevailing side-chain͞backbone paradigm. Here, the energetics of backbone hydrogen bonds dominate the folding process, with preorganization in the unfolded state. Then, under folding conditions, the resultant fold is selected from a limited repertoire of structural possibilities, each corresponding to a distinct hydrogen-bonded arrangement of ␣-helices and͞or strands of ␤-sheet.