Experimental studies of protein folding frequently are consistent with two-state folding kinetics. of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the 3C4-hairpin already is formed in the first transition state, whereas strand 1, which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts. Fyn SH3 domain, featuring the characteristic SH3 domain -sandwich fold formed by the terminal (strands 1 from Leu-3 to Ala-6 and … In practice, values for intermediate states, I, have been published for several proteins such as Im7 (12), but only very Laninamivir supplier few examples exist in the literature where values have been measured for both TS and I in studies of three-state folders (13C16), in part because of the difficulty of obtaining all four rate constants from stopped-flow-based spectroscopic approaches. By contrast, our experience with dispersion studies of SH3 domains suggests that NMR data can be analyzed to extract accurate rates (4C7, 17). NMR relaxation-dispersion-based -value analysis thus appears to be a particularly powerful method for the analysis of more Rabbit Polyclonal to CCDC45 complex folding pathways than two-state. Below we present such an analysis of the folding of the Laninamivir supplier A39V/N53P/V55L Fyn SH3 domain, which has been shown in a previous NMR study to fold by a three-state process, U ? I ? F (7). The obtained values confirm the picture of structure formation along the folding pathway that was obtained from 15N chemical shifts of the I state previously and extend it by providing information about formation of structure in the early TS, along with an indication of how structure formation evolves during the late (second) TS. The combined relaxation dispersion/-value approach facilitates the determination of complete three-state protein folding pathways at atomic detail. Methodology of -Value Analysis Before a discussion of the results of the relaxation dispersion study of A39V/N53P/V55L Fyn SH3, we briefly will review the essential features of -value analysis (1, 8C11) and its extension to three-state folding. Most experimental studies of protein folding can be interpreted by using a simple two-state model, where according to transition-state theory, the temperature dependence of the rate of transition from state X to state Y is given by the Eyring equation (18): where is a transmission coefficient, are the Boltzmann and Planck constants, respectively, and values obtained directly from the measured rates via Eq. 1. Fig. 2shows a schematic of a pair of one-dimensional energy landscapes corresponding to the folding pathways of mutant (blue) and wt (black), {along with the differences in energies attributable to mutation for each state A U, from which the A values are calculated. Fig. 2. Changes in used to calculate A (Fyn SH3) shows that Laninamivir supplier the additional aliphatic surface that accompanies the mutation, equivalent to approximately one methylene group, is solvent-exposed in F (24), and thus solvation effects should not contribute to and Fig. 2). The low TS(UI) value (13%), which is similar in magnitude to TS(UF) = ?1% obtained from a stopped-flow study of F20L Fyn SH3 (20), suggests that this position is not well structured in the first transition state. The high value for I = 73% and the intermediate value for TS(IF) = 45% are in keeping with expectations based on Laninamivir supplier a collapsed intermediate with nonnative interactions connecting the central sheet 2C3C4 with the rest of the domain that must then be broken during the second transition state, TS(IF), before formation of the native conformation. Further evidence for nonnative interactions in the intermediate is provided by the 15N chemical-shift changes in the I state that accompany the F20L mutation. The largest changes in shifts (other than at positions 20 and 21) all map to strand 1 (Leu-3, Glu-5, Ala-6, and Leu-7) (SI Figs. 8 and 9), which, as mentioned above, has been shown previously to form hydrophobic nonnative interactions in the intermediate state (7). Conversely, the largest chemical-shift changes that accompany the E5V mutation include those from Leu-18 and Ser-19. In the.