Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a Reprint requests to: Kevin W. Plaxco, Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; e-mail: kwp@chem.ucsb.edu; fax: (805) 893-4120.Abbreviations: GuHCl, guanidine hydrochloride; tris, tris hydroxymethylaminoethane; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine; CD, circular dichroism. Article published online ahead of print. Article and publication date are at

Surface charges of proteins have in several cases been found to function as "structural gatekeepers," which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the chargedepleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.folding cooperativity | protein aggregation | protein charges | protein engineering | protein folding P rotein folding is not only about optimizing the native state, but is also about avoiding misfolded traps (1-5). Such traps would otherwise compete thermodynamically with the native structures and decrease protein stability. Misfolded states that fail to properly bury "sticky" sequence material are also undesired because of their coupling to protein-aggregation disease (6-9). The general idea is that, to avoid misfolding, natural proteins have cooperative folding transitions with strong bias toward native interactions (10-13): they fold as if they were blind to alternative conformations. A clue to how this "Go-like" behavior arises is hinted by the ribosomal protein S6 (14). In essence, the S6 sequence is found to comprise certain "gatekeeper" residues (5) that block competing misfolded states by negative design (15), biasing the folding-energy landscape toward native interactions (5, 12). Mutation of these folding gatekeepers increases the propensity for S6 to misfold prior to the global folding transition in stopped-flow experiments. The phenomenon is most clearly seen in the presence of Na 2 SO 4 where the mutations induce a pronounced retardation of the refolding kinetics and characteristic roll-overs in the refolding limbs of the chevron plots (5). Notably, the chemical identity of the folding gatekeepers of S6 is not uniform but includes the buried V85, the solvent exposed E22, as well as the strain-relieving mutation A35G. The reason for this chemical diversity, as well as the detailed action of the gatekeepers, is yet not known. From a chemical standpoint it is nevertheless expected that the ubiquitous surface charges of globular proteins (16) would play a general role in negative design by their intrinsic desolvation penalties; i.e., misfolding that leads to burial ...

Site-specific information about structural heterogeneities of the protein-folding transition-state ensemble is commonly derived from the scatter of the Brønsted plot through the individual values of ‫؍‬ ⌬logkf͞⌬logKD؊N. Here, we provide a second level of site-specific detail in the transition-state analysis by demonstrating that the scatter of the Hammond plot is related to heterogeneities in the -value growth. That is, the extent of transition-state movement (⌬␤ ‡ ) is proportional to the free-energy gradient of the mutational perturbation across the top of the activation barrier, (␤ ‡ ) ؔ ⌬logKD؊N. The analysis is applied to the two-state protein L23 where the site-specific free-energy gradients are used to identify the interactions that show the highest degree of consolidation after crossing the barrier top. These interactions are distributed as a shell around the high-initiation point and denote the side-chain contacts that add criticality to the folding nucleus.transition state ͉ protein engineering ͉ -value analysis ͉ Brønsted plot ͉ Hammond behavior A major challenge in experimental studies of protein folding is to deduce more detailed, high-dimensional information about the diffuse transition-state ensemble to aid the microscopic characterization of folding nucleation and the folding free-energy landscape (1, 2). One strategy has been to see how the structural features of the transition-state ensemble change upon perturbation by denaturants and mutation. A frequently encountered example of such transition-state changes is the Hammond postulate behavior (3). The phenomenon was first reported for barnase and CI2, whose transition-state structures became more native-like as the activation free energy for unfolding was decreased by protein engineering (4, 5). Detection of the transition-state shift was by changes of the kinetic m values, i.e., by small tilts of the v-shaped chevron plots. The Hammond behavior, which has subsequently appeared as a general feature of small proteins (6-16), indicates that the top of the activation barrier is smoothly curved and displays some breadth. Upon mutational perturbation, any slanting of this curvature will cause a movement of the barrier maximum along the folding trajectory (13). From second-order polynomial curvatures of the chevron plots (4), it is further apparent that the barrier tops display an approximately quadratic curvature (13). However, the magnitude of the Hammond shifts for proteins with v-shaped chevron plots is relatively subtle and covers typically Ͻ15% of the total folding distance as measured by ␤ ‡ ϭ m f ͞(m u Ϫ m f ). Deviation from this minimal behavior is sometimes seen for proteins with pronounced distortions of the v-shaped chevron plot, indicating much larger changes of the transition state position (13, 17). The phenomenon, however, can still be accounted for by lowdimensional movements along the barrier profile, but this time in the form of discrete shifts between consecutive pointed maxima along the folding free-energy profile (7,14,(18)(19)...

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