Jeffrey K. Myers

Fast and faster: A designed variant of the B‐domain of protein A folds in 3 μsec

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B‐domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 μsec. Although wild‐type BdpA folds extremely fast, simple‐point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all‐atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence‐based time‐resolved temperature‐jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.

The Peripheral Neuropathy-Linked Trembler and Trembler-J Mutant Forms of Peripheral Myelin Protein 22 are Folding-Destabilized

Dominant mutations in the tetraspan membrane protein peripheral myelin protein 22 (PMP22) are known to result in peripheral neuropathies such as Charcot-Marie-Tooth type 1A (CMT1A) disease via mechanisms that appear to be closely linked to misfolding of PMP22 in the membrane of the endoplasmic reticulum (ER). To characterize the molecular defects in PMP22, we examined the structure and stability of two human disease mutant forms of PMP22 that are also the basis for mouse models of peripheral neuropathies: G150D ( Trembler phenotype) and L16P ( Trembler-J phenotype). Circular dichroism and NMR spectroscopic studies indicated that, when folded, the three-dimensional structures of these disease-linked mutants are similar to that of the folded wild-type protein. However, the folded forms of the mutants were observed to be destabilized relative to the wild-type protein, with the L16P mutant being particularly unstable. The rate of refolding from an unfolded state was observed to be very slow for the wild-type protein, and no refolding was observed for either mutant. These results lead to the hypothesis that ER quality control recognizes the G150D and L16P mutant forms of PMP22 as defective through mechanisms closely related to their conformational instability and/or slow folding. It was also seen that wild-type PMP22 binds Zn(II) and Cu(II) with micromolar affinity, a property that may be important to the stability and function of this protein. Zn(II) was able to rescue the stability defect of the Tr mutant.

Osmolyte effects on the self-association of concanavalin A: testing theoretical models.

The formation and stability of protein-protein interfaces are of obvious biological importance. While a large body of literature exists describing the effect of osmolytes on protein folding, very few studies address the effect of osmolytes on protein association and binding. The plant lectin concanavalin A (ConA), which undergoes a reversible tetramer-to-dimer equilibrium as a function of pH, was used as a model system to investigate the influence of nine osmolytes on protein self-association. The stabilizing or destabilizing impacts of the osmolytes were evaluated from pH titrations combined with circular dichroism spectroscopy. Relative to the dimer, trimethylamine N-oxide, betaine, proline, sarcosine, sorbitol, sucrose, and trehalose all stabilized the ConA tetramer to varying extents. Glycerol had a negligible effect, and urea destabilized the tetramer. From multiple titrations in different osmolyte concentrations, an m-value (a thermodynamic parameter describing the change in the association free energy per molar of osmolyte) was determined for each osmolyte. Experimental m-values were compared with those calculated using two theoretical models. The Tanford transfer model, with transfer free energies determined by Bolen and co-workers, failed to accurately predict the m-values in most cases. A model developed by Record and co-workers, currently applicable only to urea, betaine, and proline, more accurately predicted our experimental m-values, but significant discrepancies remained. Further theoretical work is needed to develop a thermodynamic model to predict the effect of osmolytes on protein-protein interfaces, and further experimental work is needed to determine if there is a general stabilization by osmolytes of such interfaces.