George D. Rose

Turns in Peptides and Proteins

Publisher Summary Turns are a fundamental class of polypeptide structure and are defined as sites where the peptide chain reverses its overall direction. In the past 20 years, the peptide field has witnessed major development, stimulated by the discovery of a host of bioactive peptides. Turn structures have been proposed and implicated in the bioactivity of several of these naturally occurring peptides. In addition, many structural details of turns have been derived from conformational studies of model peptides. During this same period, more than 100 complete protein structures have been elucidated in single-crystal X-ray studies. These examples document the rich diversity of structural patterns in the chain folds of native proteins. Turns are intrinsically polar structures with backbone groups that pack together closely and side chains that project outward. Such an array of atoms may constitute a site for molecular recognition, and indeed, the literature abounds with suggestions that turns serve as loci for receptor binding, antibody recognition, and post-translational modification. In peptides, turns are the conformations of choice for simultaneously optimizing both backbone–chain compactness (intramolecular nonbonded contacts) and side-chain clustering (to facilitate intermolecular recognition). Presence of turns in bioactive conformations may in fact also reflect the lack of alternative conformational possibilities. The aim of this chapter is to examine structural and functional roles of turns in peptides and proteins.

Is protein folding hierarchic? I. Local structure and peptide folding

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Nevertheless, we argue that both classes fold hierarchically and that folding begins locally. If this is the case, then the secondary structure of a protein is determined largely by local sequence information. Experimental data and theoretical considerations support this argument. Part I of this article reviews the relationship between secondary structures in proteins and their counterparts in peptides.

A molecular mechanism for osmolyte-induced protein stability

Osmolytes are small organic compounds that affect protein stability and are ubiquitous in living systems. In the equilibrium protein folding reaction, unfolded (U) ⇌ native (N), protecting osmolytes push the equilibrium toward N, whereas denaturing osmolytes push the equilibrium toward U. As yet, there is no universal molecular theory that can explain the mechanism by which osmolytes interact with the protein to affect protein stability. Here, we lay the groundwork for such a theory, starting with a key observation: the transfer free energy of protein backbone from water to a water/osmolyte solution, Δgtr, is negatively correlated with an osmolyte’s fractional polar surface area. Δgtr measures the degree to which an osmolyte stabilizes a protein. Consequently, a straightforward interpretation of this correlation implies that the interaction between the protein backbone and osmolyte polar groups is more favorable than the corresponding interaction with nonpolar groups. Such an interpretation immediately suggests the existence of a universal mechanism involving osmolyte, backbone, and water. We test this idea by using it to construct a quantitative solvation model in which backbone/solvent interaction energy is a function of interactant polarity, and the number of energetically equivalent ways of realizing a given interaction is a function of interactant surface area. Using this model, calculated Δgtr values show a strong correlation with measured values (R = 0.99). In addition, the model correctly predicts that protecting/denaturing osmolytes will be preferentially excluded/accumulated around the protein backbone. Taken together, these model-based results rationalize the dominant interactions observed in experimental studies of osmolyte-induced protein stabilization and denaturation.

Polyproline II structure in a sequence of seven alanine residues

A sequence of seven alanine residues—too short to form an α-helix and whose side chains do not interact with each other—is a particularly simple model for testing the common description of denatured proteins as structureless random coils. The 3JHNα coupling constants of individual alanine residues have been measured from 2 to 56°C by using isotopically labeled samples. The results display a thermal transition between different backbone conformations, which is confirmed by CD spectra. The NMR results suggest that polyproline II is the dominant conformation at 2°C and the content of β strand is increased by approximately 10% at 55°C relative to that at 2°C. The polyproline II conformation is consistent with recent studies of short alanine peptides, including structure prediction by ab initio quantum mechanics and solution structures for both a blocked alanine dipeptide and an alanine tripeptide. CD and other optical spectroscopies have found structure in longer “random coil” peptides and have implicated polyproline II, which is a major backbone conformation in residues within loop regions of protein structures. Our result suggests that the backbone conformational entropy in alanine peptides is considerably smaller than estimated by the random coil model. New thermodynamic data confirm this suggestion: the entropy loss on alanine helix formation is only 2.2 entropy units per residue.

Is protein folding hierarchic? II. Folding intermediates and transition states

The folding reactions of some small proteins show clear evidence of a hierarchic process, whereas others, lacking detectable intermediates, do not. Evidence from folding intermediates and transition states suggests that folding begins locally, and that the formation of native secondary structure precedes the formation of tertiary interactions, not the reverse. Some notable examples in the literature have been interpreted to the contrary. For these examples, we have simulated the local structures that form when folding begins by using the LINUS program with nonlocal interactions turned off. Our results support a hierarchic model of protein folding.

Hydrogen bonding in globular proteins

A global census of the hydrogen bonds in 42 X-ray-elucidated proteins was taken and the following demographic trends identified: (1) Most hydrogen bonds are local, i.e. between partners that are close in sequence, the primary exception being hydrogen-bonded ion pairs. (2) Most hydrogen bonds are between backbone atoms in the protein, an average of 68%. (3) All proteins studied have extensive hydrogen-bonded secondary structure, an average of 82%. (4) Almost all backbone hydrogen bonds are within single elements of secondary structure. An approximate rule of thirds applies: slightly more than one-third (37%) form i----i--3 hydrogen bonds, almost one-third (32%) form i----i--4 hydrogen bonds, and slightly less than one-third (26%) reside in paired strands of beta-sheet. The remaining 5% are not wholly within an individual helix, turn or sheet. (5) Side-chain to backbone hydrogen bonds are clustered at helix-capping positions. (6) An extensive network of hydrogen bonds is present in helices. (7) To a close approximation, the total number of hydrogen bonds is a simple function of a proteins helix and sheet content. (8) A unique quantity, termed the reduced number of hydrogen bonds, is defined as the maximum number of hydrogen bonds possible when every donor:acceptor pair is constrained to be 1:1. This quantity scales linearly with chain length, with 0.71 reduced hydrogen bond per residue. Implications of these results for pathways of protein folding are discussed.

A backbone-based theory of protein folding.

Under physiological conditions, a protein undergoes a spontaneous disorder ⇌ 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 solvent/cosolvent conditions. From the latter, we know the structures of ≈35,000 proteins, which are built on scaffolds of hydrogen-bonded structural elements, α-helix and β-sheet. Anfinsen showed that the amino acid sequence alone is sufficient to determine a proteins 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-chain/backbone 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 α-helices and/or strands of β-sheet.

Structure and Energetics of the Hydrogen-Bonded Backbone in Protein Folding

We seek to understand the link between protein thermodynamics and protein structure in molecular detail. A classical approach to this problem involves assessing changes in protein stability resulting from added cosolvents. Under any given conditions, protein molecules in aqueous buffer are in equilibrium between unfolded and folded states, U(nfolded) <==> N(ative). Addition of organic osmolytes, small uncharged compounds found throughout nature, shift this equilibrium. Urea, a denaturing osmolyte, shifts the equilibrium toward U; trimethylamine N-oxide (TMAO), a protecting osmolyte, shifts the equilibrium toward N. Using the Tanford Transfer Model, the thermodynamic response to many such osmolytes has been dissected into groupwise free energy contributions. It is found that the energetics involving backbone hydrogen bonding controls these shifts in protein stability almost entirely, with osmolyte cosolvents simply dialing between solvent-backbone versus backbone-backbone hydrogen bonds, as a function of solvent quality. This reciprocal relationship establishes the essential link between protein thermodynamics and the proteins hydrogen-bonded backbone structure.

Hierarchic organization of domains in globular proteins.

An automatic procedure is developed for the identification of domains in globular proteins from X-ray elucidated co-ordinates. Using this tool, domains are shown to be iteratively decomposable into subdomains, leading to a hierarchic molecular architecture. There is no convenient geometry that will fully characterize the atom by atom interdigitation at an interface between domains, and the strategy adopted here was devised to reduce this unwieldy three-dimensional problem to a closely approximating companion analysis in a plane. These analytically derived domain choices can be used subsequently to construct computer-generated, space-filling, color-coded views of the domains; and when this is done, the derived domains are seen to be completely resolved. The number of domains in a protein is a mathematically well-behaved function of the chain length, lending support to the supposition that the domains are an implicit structural consequence of the folding process. A spectrum of domains ranging in size from whole protein monomers to the individual units of secondary structure is apparent in each of the 22 proteins analyzed here. The hierarchic organization of structural domains is evidence in favor of an underlying protein folding process that proceeds by hierarchic condensation. In this highly constrained model, every pathway leading to the native state can be described by a tree of local folding interactions.

Polyproline II helix is the preferred conformation for unfolded polyalanine in water

Does aqueous solvent discriminate among peptide conformers? To address this question, we computed the solvation free energy of a blocked, 12‐residue polyalanyl‐peptide in explicit water and analyzed its solvent structure. The peptide was modeled in each of 4 conformers: α‐helix, antiparallel β‐strand, parallel β‐strand, and polyproline II helix (PII). Monte Carlo simulations in the canonical ensemble were performed at 300 K using the CHARMM 22 forcefield with TIP3P water. The simulations indicate that the solvation free energy of PII is favored over that of other conformers for reasons that defy conventional explanation. Specifically, in these 4 conformers, an almost perfect correlation is found between a residues solvent‐accessible surface area and the volume of its first solvent shell, but neither quantity is correlated with the observed differences in solvation free energy. Instead, solvation free energy tracks with the interaction energy between the peptide and its first‐shell water. An additional, previously unrecognized contribution involves the conformation‐dependent perturbation of first‐shell solvent organization. Unlike PII, β‐strands induce formation of entropically disfavored peptide:water bridges that order vicinal water in a manner reminiscent of the hydrophobic effect. The use of explicit water allows us to capture and characterize these dynamic water bridges that form and dissolve during our simulations. Proteins 2004.