Electric fields are fundamentally important to biological phenomena, but are difficult to measure experimentally or predict computationally. Changes in pK a of titratable residues have long been used to report on local electrostatic fields in proteins. Alternatively, nitrile vibrational probes are potentially less disruptive and more direct reporters of local electrostatic field, but quantitative interpretation is clouded by the ability of the nitrile to accept a hydrogen bond. To this end, we incorporated nitrile probes into 10 locations of staphylococcal nuclease (SNase) where pK a shifts had already been determined. We characterized the local environment of each nitrile probe experimentally, through temperature-dependent spectroscopy, and computationally, through molecular dynamics simulations, and show that hydrogen bonding interactions dominate the spectral line shapes. We demonstrate that the information provided by the line shape of the nitrile spectra, compared to scalar values of pK a shift or nitrile frequency shift, better describes local environments in proteins in a manner that will be useful for future computational efforts to predict electrostatics in complex biological systems.

Variations in backbone cleavage efficiencies during UVPD-MS of G12X variants of K-Ras are used to relate mutation identity to structural changes that impact downstream signaling with Raf.

Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational modifications, but also their tertiary structures. In this study, three metal-binding proteins, Staphylococcal nuclease, azurin, and calmodulin, are used to demonstrate the use of UVPD to elucidate metal-binding regions via comparisons between the fragmentation patterns of apo (metal-free) and holo (metal-bound) proteins. The binding of staphylococcal nuclease to calcium was evaluated, in addition to a series of lanthanide(III) ions which are expected to bind in a similar manner as calcium. On the basis of comparative analysis of the UVPD spectra, the binding region for calcium and the lanthanide ions was determined to extend from residues 40−50, aligning with the known crystal structure. Similar analysis was performed for both azurin (interrogating copper and silver binding) and calmodulin (four calcium binding sites). This work demonstrates the utility of UVPD methods for determining and analyzing the metal binding sites of a variety of classes of proteins.

Mutations of human oncoprotein p21H-Ras (hereafter “Ras”) at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. However, there is no comprehensive data set of the effects of mutations to Q61 on the protein’s intrinsic catalytic rate, structure, or interactions with water at the active site. Here, we present the first comprehensive and quantitative set of initial rates of intrinsic hydrolysis for all stable variants of RasQ61X. We further conducted enhanced molecular dynamics (MD) simulations of each construct to determine the solvent accessible surface area (SASA) of the side chain at position 61 and compared these results to previously measured changes in electric fields caused by RasQ61X mutations. For polar and negatively charged residues, we found that the rates are normally distributed about an optimal electrostatic contribution, close to that of the native Q61 residue, and the rates are strongly correlated to the number of waters in the active site. Together, these results support a mechanism of GTP hydrolysis in which Q61 stabilizes a transient hydronium ion, which then stabilizes the transition state while the γ-phosphate is undergoing nucleophilic attack by a second, catalytically active water molecule. We discuss the implications of such a mechanism on future strategies for combating Ras-based cancers.