Anthony Mittermaier

Studying excited states of proteins by NMR spectroscopy

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 °C) ground state conformation and an excited state that is 2.0 kcal mol−1 higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (ΔH = 7.1 kcal mol−1) and entropic (TΔS = 5.1 kcal mol−1, 25 °C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.

The effects of mutations on motions of side-chains in protein L studied by 2H NMR dynamics and scalar couplings.

Recently developed 2H spin relaxation experiments are applied to study the dynamics of methyl-containing side-chains in the B1 domain of protein L and in a pair of point mutants of the domain, F22L and A20V. X-ray and NMR studies of the three variants of protein L studied here establish that their structures are very similar, despite the fact that the F22L mutant is 3.2kcal/mol less stable. Measurements of methyl 2H spin relaxation rates, which probe dynamics on a picosecond-nanosecond time scale, and three-bond 3J(Cgamma-CO), 3J(Cgamma-N) and 3J(Calpha-Cdelta) scalar coupling constants, which are sensitive to motion spanning a wide range of time-scales, reveal changes in the magnitude of side-chain dynamics in response to mutation. Observed differences in the time-scale of motions between the variants have been related to changes in energetic barriers. Of interest, several of the residues with different motional properties across the variants are far from the site of mutation, suggesting the presence of long-range interactions within the protein that can be probed through studies of dynamics.

Protein stabilization by specific binding of guanidinium to a functional arginine‐binding surface on an SH3 domain

Guanidinium hydrochloride (GuHCl) at low concentrations significantly stabilizes the Fyn SH3 domain. In this work, we have demonstrated that this stabilizing effect is manifested through a dramatic (five‐ to sixfold) decrease in the unfolding rate of the domain with the folding rate being affected minimally. This behavior contrasts to the effect of NaCl, which stabilizes this domain by accelerating the folding rate. These data imply that the stabilizing effect of GuHCl is not predominantly ionic in nature. Through NMR studies, we have identified a specific binding site for guanidinium, and we have determined a dissociation constant of 90mMfor this interaction. The guanidinium‐binding site overlaps with a functionally important arginine‐binding pocket on the domain surface, and we have shown that GuHCl is a specific inhibitor of the peptide‐binding activity of the domain. A different SH3 domain possessing a similar arginine‐binding pocket is also thermodynamically stabilized by GuHCl. These data suggest that many proteins that normally interact with arginine‐containing ligands may also be able to specifically interact with guanidinium. Thus, some caution should be used when using GuHCl as a denaturant in protein folding studies. Since arginine‐mediated interactions are often important in the energetics of protein–protein interactions, our observations could be relevant for the design of small molecule inhibitors of protein–protein interactions.

The response of internal dynamics to hydrophobic core mutations in the SH3 domain from the Fyn tyrosine kinase

We have used 15N‐ and 2H‐NMR spin relaxation experiments to study the response of backbone and side‐chain dynamics when a leucine or valine is substituted for a completely buried phenylalanine residue in the SH3 domain from the Fyn tyrosine kinase. Several residues show differences in the time scales and temperature dependences of internal motions when data for the three proteins are compared. Changes were also observed in the magnitude of dynamics, with the valine, and to a lesser extent leucine mutant, showing enhanced flexibility compared to the wild‐type (WT) protein. The motions of many of the same amide and methyl groups are affected by both mutations, identifying a set of loci where dynamics are sensitive to interactions involving the targeted side chain. These results show that contacts within the hydrophobic core affect many aspects of internal mobility throughout the Fyn SH3 domain.