Beyond pKa: Experiments and Simulations of Nitrile Vibrational Probes in Staphylococcal Nuclease Show the Importance of Local Interactions.

Electric fields are fundamentally important to biological phenomena, but are difficult to measure experimentally or predict computationally. Changes in pKa 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 pKa 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 pKa 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.

Quantifying the Extent of Hydration of a Surface-Bound Peptide Using Neutron Reflectometry.

Establishing how water, or the absence of water, affects the structure, dynamics, and function of proteins in contact with inorganic surfaces is critical to developing successful protein immobilization strategies. In the present article, the quantity of water hydrating a monolayer of helical peptides covalently attached to self-assembled monolayers (SAMs) of alkyl thiols on Au was measured using neutron reflectometry (NR). The peptide sequence was composed of repeating LLKK units in which the leucines were aligned to face the SAM. When immersed in water, NR measured 2.7 ± 0.9 water molecules per thiol in the SAM layer and between 75 ± 13 and 111 ± 13 waters around each peptide. The quantity of water in the SAM was nearly twice that measured prior to peptide functionalization, suggesting that the peptide disrupted the structure of the SAM. To identify the location of water molecules around the peptide, we compared our NR data to previously published molecular dynamics simulations of the same peptide on a hydrophobic SAM in water, revealing that 49 ± 5 of 95 ± 8 total nearby water molecules were directly hydrogen-bound to the peptide. Finally, we show that immersing the peptide in water compressed its structure into the SAM surface. Together, these results demonstrate that there is sufficient water to fully hydrate a surface-bound peptide even at hydrophobic interfaces. Given the critical role that water plays in biomolecular structure and function, these results are expected to be informative for a broad array of applications involving proteins at the bio/abio interface.

Agreement between Experimental and Simulated Circular Dichroic Spectra of a Positively Charged Peptide in Aqueous Solution and on Self-Assembled Monolayers.

Successfully immobilizing functional proteins on inorganic surfaces has long been a challenge to the biophysics and bioengineering communities. This is due, in part, to a lack of understanding of the effect of nonaqueous environments on protein structure from both experimental and computational perspectives. Because most experimental information about protein structure comes from the Protein Data Bank and is collected from an aqueous solvent environment, modern force fields for molecular dynamics (MD) simulations are parameterized against these data. The applicability of such force fields to biomolecules in different environments, including when in contact with surfaces and substrates, must be validated. Here, we present MD folding simulations of a highly charged peptide solvated in water, solvated in a solution of 2:1 t-BuOH/H2O and bound to the surface of a methyl-terminated self-assembled monolayer (SAM), and compare the structures predicted by these simulations to previously reported circular dichroism spectra. We show quantitative agreement between experiments and simulations of solvent- and surface-induced conformational changes of a positively charged peptide in these three environments. We show further that the surface-bound peptide must fold before chemically reacting with the surface. Finally, we demonstrate that a well-ordered SAM is critical to the folding process. These results will guide further simulations of peptides and proteins in diverse and complex environments.

Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras.

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.

Quantifying the Effects of Hydrogen Bonding on Nitrile Frequencies in GFP: Beyond Solvent Exposure.

Vibrational spectroscopy is a powerful tool for characterizing the complex noncovalent interactions that arise in biological systems. The nitrile stretching frequency has proven to be a particularly convenient biological probe, but the interpretation of nitrile spectroscopy is complicated by its sensitivity to local hydrogen bonding interactions. This often inhibits the straightforward interpretation of nitrile spectra by requiring knowledge of the molecular-level details of the local environment surrounding the probe. While the effect of hydrogen bonds on nitrile frequencies has been well-documented for small molecules in solution, there have been relatively few studies of these effects in a complex protein system. To address this, we introduced a nitrile probe at nine locations throughout green fluorescent protein (GFP) and compared the mean vibrational frequency of each probe to the specific hydrogen bonding geometries observed in molecular dynamics (MD) simulations. We show that a continuum of hydrogen bonding angles is found depending on the particular location of each nitrile, and that the differences in these angles account for the differences in the measured nitrile frequency. We further observed that the temperature dependence of the nitrile frequencies (measured as a frequency-temperature line slope, FTLS) was a good indicator of the hydrogen bonding interactions of the probe, even in scenarios where the nitrile was involved in complex and restricted hydrogen bonds, both from protein and from water. While constant offsets to the nitrile frequency to all hydrogen bonding environments have been applied before to interpret shifts in nitrile frequency, we show that this is insufficient in systems where the hydrogen bonds may be restricted by the surrounding medium. However, the strength of the observed correlation between nitrile frequency and hydrogen bonding angle suggests that it may be possible to disentangle electrostatic effects and effects of the orientation of hydrogen bonding on the nitrile stretching frequency. Meanwhile, the experimental measurement of the FTLS of the nitrile is an excellent tool to identify changes in the hydrogen bonding interactions for a particular probe.

Orthogonal Electric Field Measurements near the Green Fluorescent Protein Fluorophore through Stark Effect Spectroscopy and pKa Shifts Provide a Unique Benchmark for Electrostatics Models.

Measurement of the magnitude, direction, and functional importance of electric fields in biomolecules has been a long-standing experimental challenge. pKa shifts of titratable residues have been the most widely implemented measurements of the local electrostatic environment around the labile proton, and experimental data sets of pKa shifts in a variety of systems have been used to test and refine computational prediction capabilities of protein electrostatic fields. A more direct and increasingly popular technique to measure electric fields in proteins is Stark effect spectroscopy, where the change in absorption energy of a chromophore relative to a reference state is related to the change in electric field felt by the chromophore. While there are merits to both of these methods and they are both reporters of local electrostatic environment, they are fundamentally different measurements, and to our knowledge there has been no direct comparison of these two approaches in a single protein. We have recently demonstrated that green fluorescent protein (GFP) is an ideal model system for measuring changes in electric fields in a protein interior caused by amino acid mutations using both electronic and vibrational Stark effect chromophores. Here we report the changes in pKa of the GFP fluorophore in response to the same mutations and show that they are in excellent agreement with Stark effect measurements. This agreement in the results of orthogonal experiments reinforces our confidence in the experimental results of both Stark effect and pKa measurements and provides an excellent target data set to benchmark diverse protein electrostatics calculations. We used this experimental data set to test the pKa prediction ability of the adaptive Poisson-Boltzmann solver (APBS) and found that a simple continuum dielectric model of the GFP interior is insufficient to accurately capture the measured pKa and Stark effect shifts. We discuss some of the limitations of this continuum-based model in this system and offer this experimentally self-consistent data set as a target benchmark for electrostatics models, which could allow for a more rigorous test of pKa prediction techniques due to the unique environment of the water-filled GFP barrel compared to traditional globular proteins.