Stereoelectronic and steric effects in side chains preorganize a protein main chain

Abstract

Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in ðProProGlyÞ 7 collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T m values that are increased by >50°C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 Å reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2∕3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.collagen triple helix | nonnatural amino acid | preorganization | protein stability | x-ray crystallography T he three-dimensional structures of proteins are stable because of a delicate balance of forces (1). Increases in the conformational stability of a protein-desirable in many contexts (2)-can be realized by enhancing the hydrophobic effect (3, 4), introducing or shielding a hydrogen bond (5) or electrostatic interaction (6-8), or introducing a disulfide crosslink (9-11) or metal ion-binding site (12). These strategies are often frustrated by enthalpy-entropy compensation, whereby enthalpic stabilization is offset entirely by entropic destabilization, or vice versa (13). Moreover, the strategies often lack subtlety and can lead to a misshapen and hence dysfunctional protein.As elaborated by Cram (14), the principle of preorganization states that, "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes." In a typical manifestation, a conformational constraint is imposed upon a receptor or ligand during its chemical synthesis. This principle can also yield biopolymers with increased conformational stability (Fig. 1). For example, the stability of a DNA duplex correlates with the helicity of its single strands (15, 16), and a bicyclic ("locked") derivative of ribose enhances that stability (17, 18). The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21-23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24-27).We suspected that alterations to proline residues could provide a particularly incisive means to preorganize the conformation of a polypeptide chain. In classic work, Matthews and coworkers substituted proline, which is the least flexible residu...

Stereochemical Determinants of C-terminal Specificity in PDZ Peptide-binding Domains

“…However, when both conformers were modeled, the alternative conformation was refined to an occupancy value Յ20%. Although a proline pucker can influence main-chain orientation (46), in this case, the effects on neighboring residues are on the scale of the maximum likelihood coordinate error (0.16 Å). Thus, our final model includes only the dominant P Ϫ4 conformation.…”

Interstrand Dipole-Dipole Interactions Can Stabilize the Collagen Triple Helix

“…We used a heating rate of 0.1°C/min for these scans (10,32), finding that slower rates increased the noise substantially. Subsequent scans of the same sample showed high reversibility, with Ͼ85% endotherm recovery upon re-equilibration at 4°C.…”

“…Still, we were skeptical that the hyperstability of (HypHypGly) n triple helices is due to the release of water upon triple-helix folding and hydroxyl group burial. We had shown previously that attributing differences in thermostability to hydration in the context of the collagen triple helix can be problematic (5,7,10,38,39).…”

Computational design of self-assembling register-specific collagen heterotrimers

“…We set the prototypical homotrimeric sequence, (POG) 10 , as the reference state and gave its stability a numerical value of 0 in our relative scale. Single point mutations with respect to this scaffold, which are known to be destabilizing 13 , are given a positive numerical value.…”

“…P and O have a preference for distinct conformations of the pyrrolidine side chain that biases the main chain φ dihedrals to values close to those found in the X and Y positions of the triple helix, thus reducing the unfavourable conformational entropy change on the assembly of the unfolded chains 10 . The glycine residues, present at every third position in the sequence, pack tightly in the core of the helix forcing the peptide chains to self-assemble with a one amino-acid stagger between adjacent strands.…”