Tryptophan stabilizes His-heme loops in the denatured state only when it is near a loop end.

It is long accepted that a protein which folds to a well-defined tertiary structure encodes its structural preference in its primary structure (1, 2). Furthermore, theoretical work indicates that foldable protein sequences have evolved to provide minimally frustrated energy landscapes (3, 4). Thus, primary sequence encodes not only structure, but an efficient folding mechanism. Given this context, it is somewhat surprising that denatured proteins have scaling properties consistent with a classic random coil (5). The resulting viewpoint that the denatured state ensemble (DSE)1 is devoid of residual structure of substantive import has been reinforced by observations that polypeptides with simple (and unfoldable) sequences have random coil scaling properties (6–8). Theoretical studies, however, demonstrate that random coil scaling can be consistent with substantial residual structure (9). We have recently shown with our His-heme loop formation method that the scaling properties of simple alanine-rich sequences and foldable sequences are similar (10). However, whereas the alanine-rich sequences adhere strictly to random coil scaling, the local variation in chain properties for a foldable heteropolymer is substantial (10, 11). Considerable evidence has accrued, primarily from NMR structural methods and molecular dynamics (MD) simulations, that denatured states retain residual structure (12–14), both native and non-native in nature (15–24). Substantial data also show that residual structure can involve long-range tertiary interactions (17, 18, 25–32), even under strongly denaturing conditions (30). However, there are counter-examples where local clusters of residual structure have no detectable long-range interactions with other clusters (33, 34). Aromatic residues, particularly tryptophan (15, 16, 19, 22, 29, 30, 35–39), appear to promote residual structure in the DSE. Despite the plethora of structural data, thermodynamic data on the stability of residual structure in the DSE is scarce (12, 40), but important for understanding the role of residual structure in the DSE in specifying tertiary structure and limiting the search to find the native tertiary structure of a protein. We have recently developed a host-guest variation of our His-heme loop formation method which has provided some of the first quantitative thermodynamic data on the tendency of different amino acids to stabilize residual structure in a denatured protein (41). Since the relative stability of simple loops will be important for defining the topology of a fold early in folding, quantifying the strength of residual structure interactions in the DSE and the distance over which these interactions operate is essential. The site of the guest residue in our initial host-guest study was five residues from one end of the loop (41). Thus, the primary source of the observed stabilization of the loop could be from interactions between residues near the loop ends, since the residues near the ends of the loop (histidine and heme in our system, see Figure 1) are held proximal to each other when the loop is formed. However, it is possible that the reduced conformational space in the closed loop form could promote longer-range interactions between residues within the loop even if they are not near the ends of the loop (42, 43). Figure 1 Schematic representation of denatured state His-heme loop formation with the 28-residue loop containing a 12-residue alanine-rich host sequence. The residues of the alanine-rich insert between His(–2) and Ala(-1) of iso-1-Cytc are represented ... In the present study, we apply our host-guest approach with a variant of yeast iso-1-cytochrome c (iso-1-Cytc) that has a 12-residue alanine-rich sequence engineered into the N-terminus of the protein (the host variant, NH5A-2, see Figure 1). The NH5A-2 variant can form a 28-residue His-heme loop when the protein is in the denatured state. We use a set of guest variants to compare the effect on the stability of the 28-residue denatured state His-heme loop when an Ala residue is replaced with a guest Trp residue 4 positions from the His end of the loop (AW(4) variant, see Figure 1) with the effect on the stability of the 28-residue denatured state His-heme loop when an Ala residue is replaced with a guest Trp residue 10 positions from the His end of the loop (AW(10) variant, see Figure 1). We also introduce the two Trp guest residues simultaneously (AW(4,10) variant, see Figure 1). When a disordered polypeptide chain forms a loop, the residues within the loop have less conformational freedom. This loss in conformational freedom can be viewed as an increase in the local concentration of the residues within the loop with respect to each other. The increase in the local concentration of the residues within the loop might be expected to promote interactions between residues within the loop through a mass action effect (Le Chatelier’s principle) when the loop forms (42, 43). The effect of increased local concentration when the loop forms should be greater near the ends of the loop than near the center of the loop, since residues at the ends of the loops have more nearest neighbors after the loop forms (see Figure 1). Thus, depending on the sequence distance over which residue-residue interactions occur, the effect of replacing Ala with the more hydrophobic residue Trp might be expected to be greater when it is placed near the end of the loop (AW(4) variant) versus further from the end of the loop (AW(10) variant). Since solvent quality has significant effects on denatured state properties (44–46), we have made measurements of His-heme loop formation over the range 1.5 to 6.0 M guanidine hydrochloride (GdnHCl) to determine how solvent quality affects the thermodynamics and kinetics of residual structure stabilization by Trp relative to Ala in the denatured state. We find that the 28-residue denatured state His-heme loop is stabilized and that this stabilization increases with decreasing GdnHCl concentration when Trp guest is 4-residues, but not when it is 10-residues, from the loop end.