Integrating the function of biological molecules into traditional inorganic materials and substrates couples biologically relevant function to synthetic devices and generates new materials and capabilities by combining biological and inorganic functions. At this so-called “bio/abio interface,” basic biological functions such as ligand binding and catalysis can be co-opted to detect analytes with exceptional sensitivity or to generate useful molecules with chiral specificity under entirely benign reaction conditions. Proteins function in dynamic, complex, and crowded environments (the living cell) and are therefore appropriate for integrating into multistep, multiscale, multimaterial devices such as integrated circuits and heterogeneous catalysts. However, the goal of reproducing the highly specific activities of biomolecules in the perturbed chemical and electrostatic environment at an inorganic interface while maintaining their native conformations is challenging to achieve. Moreover, characterizing protein structure and function at a surface is often difficult, particularly if one wishes to compare the activity of the protein to that of the dilute, aqueous solution phase. Our laboratory has developed a general strategy to address this challenge by taking advantage of the structural and chemical properties of alkanethiol self-assembled monolayers (SAMs) on gold surfaces that are functionalized with covalently tethered peptides. These surface-bound peptides then act as the chemical recognition element for a target protein, generating a biomimetic surface in which protein orientation, structure, density, and function are controlled and variable. Herein we discuss current research and future directions related to generating a chemically tunable biofunctionalization strategy that has potential to successfully incorporate the highly specialized functions of proteins onto inorganic substrates.

A photoactivatable variant of superfolder green fluorescent protein (GFP) was created by replacing the threonine at position 203 with aspartic acid. Photoactivation by exposure of this mutant to UV light resulted in conversion of the fluorophore from the neutral to the negatively charged form, accompanied by a ∼95-fold increase in fluorescence under 488 nm excitation. Mass spectrometry before and after exposure to UV light revealed a change in mass of 88 Da, attributed to the double decarboxylation of Glu 222 and Asp 203. Kinetics studies and nonlinear power-dependence of the initial rate of photoconversion indicated that the double decarboxylation occurred via a multiphoton absorption process at 254 nm. In addition to providing a photoactivatable GFP with robust folding properties, a detailed mechanistic understanding of this double decarboxylation in GFP will lead to a better understanding of charge transfer in fluorescent proteins.

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.

There is growing interest in using the nitrile vibrational oscillation as a site-specific probe of local environment to study dynamics, folding, and electrostatics in biological molecules such as proteins. Nitrile probes have been used extensively as reporters of electric field using vibrational Stark effect spectroscopy. However, the analysis of frequencies in terms of electric fields is potentially complicated by the large ground state dipole moment of the nitrile, which may irrevocably perturb the protein under investigation, and the ability of nitriles to accept hydrogen bonds, which causes frequency shifts that are not described by the Stark effect. The consequence of this is that vibrational spectroscopy of nitriles in biomolecules could be predominately sensitive to their local hydration status, not electrostatic environment, and have the potential to be particularly destabilizing to the protein. Here, we introduce green fluorescent protein (GFP) as a model system for addressing these concerns using biosynthetically incorporated p-cyanophenylalanine (pCNF) residues in the interior of GFP and measuring absorption energies of both the intrinsic GFP fluorophore and pCNF residues in response to a series of amino acid mutations. We show that observed changes in emission energy of GFP due to the mutations strongly correlate with changes in electric field experienced by both the nitrile probes and the intrinsic fluorophore. Additionally, we show that changes in electric field measured from the intrinsic fluorophore due to amino acid mutations are unperturbed by the addition of pCNF residues inserted nearby. Finally, we show that changes in electric field experienced by the vibrational probes trend monotonically with changes in field experienced by the native fluorophore even though the nitrile probe is engaged in moderate hydrogen bonding to nearby water molecules, indicated by the temperature dependence of the nitrile’s absorption energy. Together these results demonstrate that even in the presence of hydrogen bonding it is possible to relate nitrile absorption frequencies to electrostatic environment by comparing highly similar environments. GFP’s intrinsic linear sensitivity to electric fields makes it a convenient model system for studying electrostatics in proteins that offers lessons for proteins without this visible fluorophore.

Fluoroethylene carbonate (FEC) has become a standard electrolyte additive for use with silicon negative electrodes, but how FEC affects solid electrolyte interphase (SEI) formation on the silicon anode’s surface is still not well understood. Herein, SEI formed from LiPF6-based carbonate electrolytes, with and without FEC, were investigated on 50 nm thick amorphous silicon thin film electrodes to understand the role of FEC on silicon electrode surface reactions. In contrast to previous work, anhydrous and anoxic techniques were used to prevent air and moisture contamination of prepared SEI films. This allowed for accurate characterization of the SEI structure and composition by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry depth profiling. These results show that FEC reduction leads to fluoride ion and LiF formation, consistent with previous computational and experimental results. Surprisingly, we also find that these species decrease lithium-ion solubility and increase the reactivity of the silicon surface. We conclude that the effectiveness of FEC at improving the Coulombic efficiency and capacity retention is due to fluoride ion formation from reduction of the electrolyte, which leads to the chemical attack of any silicon-oxide surface passivation layers and the formation of a kinetically stable SEI comprising predominately lithium fluoride and lithium oxide.

We investigate the source of Raman background signal commonly misidentified as fluorescence in nonaqueous electrolytes via a variety of spectroscopies (Raman, fluorescence, NMR) and find evidence of hydrogen-bonding interactions. This hydrogen bonding gives rise to broadband anharmonic vibrational modes and suggests that anions play an important and underappreciated role in the structure of nonaqueous electrolytes. Controlling electrolyte structure has important applications in advancing in operando spectroscopy measurements as well as understanding the stability of high concentration electrolytes for next-generation electrochemical energy storage devices.

Biological function emerges in large part from the interactions of biomacromolecules in the complex and dynamic environment of the living cell. For this reason, macromolecular interactions in biological systems are now a major focus of interest throughout the biochemical and biophysical communities. The affinity and specificity of macromolecular interactions are the result of both structural and electrostatic factors. Significant advances have been made in characterizing structural features of stable protein–protein interfaces through the techniques of modern structural biology, but much less is understood about how electrostatic factors promote and stabilize specific functional macromolecular interactions over all possible choices presented to a given molecule in a crowded environment. In this Feature Article, we describe how vibrational Stark effect (VSE) spectroscopy is being applied to measure electrostatic fields at protein–protein interfaces, focusing on measurements of guanosine triphosphate (GTP)-binding proteins of the Ras superfamily binding with structurally related but functionally distinct downstream effector proteins. In VSE spectroscopy, spectral shifts of a probe oscillator’s energy are related directly to that probe’s local electrostatic environment. By performing this experiment repeatedly throughout a protein–protein interface, an experimental map of measured electrostatic fields generated at that interface is determined. These data can be used to rationalize selective binding of similarly structured proteins in both in vitro and in vivo environments. Furthermore, these data can be used to compare to computational predictions of electrostatic fields to explore the level of simulation detail that is necessary to accurately predict our experimental findings.

The ability to maintain or reproduce biomolecular structures on inorganic substrates has the potential to impact diverse fields such as sensing and molecular electronics, as well as the study of biological self-assembly and structure–function relationships. Because the structure and self-assembly of biomolecules are exquisitely sensitive to their local chemical and electrostatic environment, the goal of reproducing or mimicking biological function in an abiological environment, including at a surface, is challenging. However, simple and well-characterized chemical modifications of prepared surfaces can be used to tune surface chemistry, structure, electrostatics, and reactivity of inorganic materials to facilitate biofunctionalization and function. Here, we describe the covalent attachment of 13-residue β-stranded peptides containing alkyne groups to a flat gold surface functionalized with an azide-terminated self-assembled monolayer through a Huisgen cycloaddition, or “click”, reaction. The chemical composition and structural morphology of these surfaces were characterized using X-ray photoelectron spectroscopy, grazing incidence angle reflection–absorption infrared spectroscopy, surface circular dichroism, and atomic force microscopy. The surface-bound β-strands self-assemble into antiparallel β-sheets to form fibrillar structures 24.9 ± 1.6 nm in diameter and 2.83 ± 0.74 nm in height on the reactive surface. The results herein provide a platform for studying and controlling the self-assembly process of biomolecules into larger supermolecular structures while allowing tunable control through chemical functionalization of the surface. Interest in the mechanisms of formation of fibrillar structures has most commonly been associated with neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, but fibrils may actually represent the thermodynamic low-energy conformation of a much larger class of peptides and proteins. The protocol developed here is an important step toward uncovering not only the factors that dictate self-assembly but also the mechanisms by which this fibrillar class of superstructures forms.

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the headgroup region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction of the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped toward the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ∼3 cm–1 greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in an absolute magnitude of 8–11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed.

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras–Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras–Raf and Ras–RalGDS complexes.

We have examined the effects of including explicit, near-probe solvent molecules in a continuum electrostatics strategy using the linear Poisson–Boltzmann equation with the Adaptive Poisson–Boltzmann Solver (APBS) to calculate electric fields at the midpoint of a nitrile bond both at the surface of a monomeric protein and when docked at a protein–protein interface. Results were compared to experimental vibrational absorption energy measurements of the nitrile oscillator. We examined three methods for selecting explicit water molecules: (1) all water molecules within 5 Å of the nitrile nitrogen; (2) the water molecule closest to the nitrile nitrogen; and (3) any single water molecule hydrogen-bonding to the nitrile. The correlation between absolute field strengths with experimental absorption energies were calculated and it was observed that method 1 was only an improvement for the monomer calculations, while methods 2 and 3 were not significantly different from the purely implicit solvent calculations for all protein systems examined. Upon taking the difference in calculated electrostatic fields and comparing to the difference in absorption frequencies, we typically observed an increase in experimental correlation for all methods, with method 1 showing the largest gain, likely due to the improved absolute monomer correlations using that method. These results suggest that, unlike with quantum mechanical methods, when calculating absolute fields using entirely classical models, implicit solvent is typically sufficient and additional work to identify hydrogen-bonding or nearest waters does not significantly impact the results. Although we observed that a sphere of solvent near the field of interest improved results for relative field calculations, it should not be consider a panacea for all situations.

The introduction of scanning probe microscopy (SPM) techniques revolutionized the field of condensed matter science by allowing researchers to probe the structure and composition of materials on an atomic scale. Although these methods have been used to make molecular- and atomic-scale measurements on biological systems with some success, the biophysical sciences remain on the cusp of a breakthrough with SPM technologies similar in magnitude to that experienced by fields related to solid-state surfaces and interfaces. Numerous challenges arise when attempting to connect biological molecules that are often delicate, dynamic, and complex with the experimental requirements of SPM techniques. However, there are a growing number of studies in which SPM has been successfully used to achieve subnanometer resolution measurements in biological systems where carefully designed and prepared samples have been paired with appropriate SPM techniques. We review significant recent innovations in applying SPM techniques to biological molecules, and highlight challenges that face researchers attempting to gain atomic- and molecular-level information of complex biomolecular structures.Keywords: AFM; DNA; lipid bilayer; NSOM; protein; scanning probe microscopy; STM;

Electrostatic fields at the interface of the GTPase H-Ras (Ras) docked with the Ras binding domain of the protein Ral guanine nucleoside dissociation stimulator (Ral) were measured with vibrational Stark effect (VSE) spectroscopy. Nine residues on the surface of Ras that participate in the protein–protein interface were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe into the protein–protein interface. The absorption energy of the nitrile was measured both on the surface of Ras in its monomeric state, then after incubation with the Ras binding domain of Ral to form the docked complex. Boltzmann-weighted structural snapshots of the nitrile-labeled Ras protein were generated both in monomeric and docked configurations from molecular dynamics simulations using enhanced sampling of the cyanocysteine side chain's χ2 dihedral angle. These snapshots were used to determine that on average, most of the nitrile probes were aligned along the Ras surface, parallel to the Ras–Ral interface. The average solvent-accessible surface areas (SASA) of the cyanocysteine side chain were found to be <60 Å2 for all measured residues, and was not significantly different whether the nitrile was on the surface of the Ras monomer or immersed in the docked complex. Changes in the absorption energy of the nitrile probe at nine positions along the Ras–Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and found a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface. These data are used to rationalize several puzzling features of the Ras–Ral interface.

Continuum electrostatics methods are commonly used to calculate electrostatic potentials in proteins and at protein–protein interfaces to aid many types of biophysical studies. Despite their ubiquity throughout the biophysical literature, these calculations are difficult to test against experimental data to determine their accuracy and validity. To address this, we have calculated the Boltzmann-weighted electrostatic field at the midpoint of a nitrile bond placed at a variety of locations on the surface of the protein RalGDS, both in its monomeric form as well as when docked to four different constructs of the protein Rap, and compared the computation results to vibrational absorption energy measurements of the nitrile oscillator. This was done by generating a statistical ensemble of protein structures using enhanced molecular dynamics sampling with the Amber03 force field, followed by solving the linear Poisson–Boltzmann equation for each structure using the Applied Poisson–Boltzmann Solver (APBS) software package. Using a two-stage focusing strategy, we examined numerous second stage box dimensions, grid point densities, box locations, and compared the numerical result to the result obtained from the sum of the numeric reaction field and the analytic Coulomb field. It was found that the reaction field method yielded higher correlation with experiment for the absolute calculation of fields, while the numeric solutions yielded higher correlation with experiment for the relative field calculations. Finer grid spacing typically improved the calculation, although this effect was less pronounced in the reaction field method. These sorts of calculations were also very sensitive to the box location, particularly for the numeric calculations of absolute fields using a 103 Å3 box.

Peptide-terminated monolayers were formed through a Huisgen cycloaddition reaction between an α-helical peptide containing two propargylglycine unnatural functional groups 20 Å apart and an alkanethiol self-assembled monolayer (SAM) on a gold surface containing 25% surface density of reactive azide terminal groups. The azide- and peptide-terminated surfaces were imaged by scanning tunneling microscopy (STM) using a low tunneling current of 10 pA. On the peptide-terminated surface, oblong features ∼30 Å long and ∼20 Å wide were observed and attributed to individual surface-bound α-helical peptides oriented parallel to the gold surface. These features covered an area of the surface corresponding to a density of 0.11 ± 0.01 peptides nm–2, compared with a theoretical density of ∼0.14 peptides nm–2 for a fully reacted surface. Finally, no evidence of peptide aggregation was observed on either short (<10 nm) or long (∼100 nm) length scales.

Mutations of human oncoprotein p21Ras (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 electrostatically stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. We have tested the interactions between amino acids at this position and water by measuring changes in the electrostatic field experienced by a nitrile probe positioned near Ras Q61 using vibrational Stark effect (VSE) spectroscopy. We mutated this glutamine to every amino acid except cysteine and proline and then incubated these mutants with a Ral guanine nucleotide dissociation stimulator (Ral) containing the I18C mutation that was chemically labeled with a thiocyanate vibrational spectroscopic probe. The formation of the docked Ras Q61X–labeled Ral complex was confirmed by measurement of the dissociation constant of the interaction. We measured the absorption energy of this nitrile to determine any differences in electrostatic environment in the immediate vicinity of the thiocyanate probe between wild type and mutants of Ras. For each Ras Q61X mutant, we correlate the change in electrostatic field at position 61 with the solvent accessible surface area of polar components of the mutant side chain determined from a Boltzmann-weighted ensemble of structures, as well as the residue’s hydration potential. These results support the hypothesis that the role of Ras Q61 is to stabilize water in or near the active site during GTP hydrolysis. The substantial effect that nonpolar side chains of Ras Q61X have on the absorption energy of the thiocyanate must be investigated with further experiments.

The human protein Rap1A (Rap) is a member of the Ras superfamily of GTPases that binds to the downstream effector Ral guanine nucleotide dissociation stimulator (RalGDS). Although Ras and Rap have nearly identical amino acid sequences and structures along the effector binding surface, the charge reversal mutation Rap K31E has previously been shown to increase the dissociation constant of Rap-RalGDS docking to values similar to that of Ras-RalGDS docking. This indicates that the difference in charge at position 31 could provide a mechanism for Ral to distinguish two structurally similar but functionally distinct GTPases, which would be of vital importance for appropriate biological function. In this report, vibrational Stark effect spectroscopy, dissociation constant measurements, and molecular dynamics simulations were used to investigate the role that electrostatic field differences caused by the charge reversal mutation Rap K31E play in determining the binding specificity of RalGDS to Rap versus Ras. To do this, six variants of RalGDS carrying a thiocyanate electrostatic probe were docked with three Rap mutants, E30D, K31E, and E30D/K31E. The change in absorption energy of the thiocyanate probe caused by RalGDS docking to these Rap variants was then compared to that observed with wild-type Ras. Three trends emerged: the expected reversion behavior, an additive behavior of the two single mutations, and cancelation of the effects of each single mutation in the double mutant. These observations are explained with a physical model of the position of the thiocyanate probe with respect to the mutated residue based on molecular dynamics simulations.

Since the potential for alloying lithium with silicon is outside the window of stability of common commercial electrolytes, silicon surfaces form an amorphous solid electrolyte interphase (SEI) under applied potential, which hampers silicon's performance as a lithium-ion battery anode. We have investigated the composition, distribution, and ambient stability of the SEI formed on undoped silicon (001) wafers configured as model electrodes in three different electrochemical conditions using a reduced oxidation interface for transporting air-sensitive samples from a glovebox to an ultra-high-vacuum chamber for X-ray photoelectron spectroscopy (XPS) analysis. Variable potential cycling and step experiments included linear sweep voltammetry (LSV), cyclic voltammetry (CV), and chronoamperometry (CA). CV and LSV experiments on silicon electrodes scanned from open-circuit voltage to lithiation (3–0.01 V vs Li/Li+) showed a suppression of carbonate-containing species relative to CA experiments (potential step for 300 s at 0.01 V vs Li/Li+) in anoxic XPS measurements. When silicon electrodes were exposed to ambient air, SEI layers reacted through both fluorination and combustion processes to produce different SEI product distributions than those prepared under anoxic conditions.

Gold and quartz surfaces terminated in an alkane thiol self-assembled monolayer (SAM) that were partially terminated with azide were reacted with a helical peptide containing two alkyne groups in a Cu(I)-catalyzed Huisgen cycloaddition. Surface grazing incidence angle reflection–absorption infrared spectroscopy (GRAS-IR) was used to determine that when the Au surface was terminated with 25% of the monolayer containing azide groups, 92% of available azide groups reacted with the peptide. The majority of peptides reacted with both alkynes, resulting in peptides tethered to the surface through two covalent bonds. This was confirmed by comparison to a control peptide containing only one reactive alkyne group. Surface circular dichroic (CD) spectroscopy showed that while the helical structure of the peptide was distorted in the reaction solution, α-helical structure was induced when tethered on the SAM functionalized Au surface. Demonstration of the preservation of desired secondary structure of helical elements at a chemically functionalized surface is an important advance in preparing robust biologically mimetic surfaces to integrate functioning proteins into inorganic materials.

Electrostatic fields in lipid bilayer membranes influence the structure and function of membrane-associated proteins. We present here the first direct measurement of the membrane dipole electrostatic field in lipid bicelles using vibrational Stark effect spectroscopy, in which a nitrile oscillator’s vibrational frequency changes in response to its local electrostatic environment. We synthesized α-helical peptides containing the unnatural amino acid p-cyanophenylalanine (CN-Phe) at four locations along the helix. This peptide was intercalated into bicelles 5 and 15 nm in radius composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). Changes in the vibrational absorption energy of the nitrile probe at each position along the helical axis were used to determine changes in the local electrostatic field of the probe. We measured the magnitude of the membrane dipole electrostatic field to be −6 MV/cm, changing rapidly near the membrane surface and more slowly in the low dielectric membrane interior.Keywords: bicelle; DHPC; DMPC; membrane dipole potential; membrane electrostatic field; p-cyanophenylalanine; vibrational stark effect;

Electrostatic fields at the interface of the Ras binding domain of the protein Ral guanine nucleotide dissociation stimulator (RalGDS) with the structurally analogous GTPases Ras and Rap1A were measured with vibrational Stark effect (VSE) spectroscopy. Eleven residues on the surface of RalGDS that participate in this protein−protein interaction were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe in the form of the thiocyanate (SCN) functional group. The measured SCN absorption energy on the monomeric protein was compared with solvent-accessible surface area (SASA) calculations and solutions to the Poisson−Boltzmann equation using Boltzmann-weighted structural snapshots from molecular dynamics simulations. We found a weak negative correlation between SASA and measured absorption energy, indicating that water exposure of protein surface amino acids can be estimated from experimental measurement of the magnitude of the thiocyanate absorption energy. We found no correlation between calculated field and measured absorption energy. These results highlight the complex structural and electrostatic nature of the protein−water interface. The SCN-labeled RalGDS was incubated with either wild-type Ras or wild-type Rap1A, and the formation of the docked complex was confirmed by measurement of the dissociation constant of the interaction. The change in absorption energy of the thiocyanate functional group due to complex formation was related to the change in electrostatic field experienced by the nitrile functional group when the protein−protein interface forms. At some locations, the nitrile experiences the same shift in field when bound to Ras and Rap1A, but at others, the change in field is dramatically different. These differences identify residues on the surface of RalGDS that direct the specificity of RalGDS binding to its in vivo binding partner, Rap1A, through an electrostatic mechanism.

Gold surfaces functionalized with an α-helical peptide have been generated by reacting an azide-terminated self-assembled monolayer with structured peptides containing two cyanophenylalanines through a Huisgen cycloaddition. Mixed monolayers of a reactive bromine-terminated thiol and inert alkane thiol were prepared at various concentrations of the Br-terminated moiety. These were reacted with sodium azide to form azide-terminated monolayers with controlled concentration of the reactive azide. These surfaces were studied through ellipsometry and X-ray photoelectron spectroscopy, which demonstrated that the concentration of the reactive azide group on the surface is controlled by the chemical conditions under which the monolayer is prepared. Grazing incident angle surface infrared spectroscopy (GRAS-IR) of the azide-terminated surface demonstrated that the azide is approximately perpendicular to the plane of the surface, as expected. These surfaces were then exposed to an α-helical peptide composed of alternating leucine and lysine residues, with two residues replaced with cyanophenylalanine to react with two neighboring surface-bound azide groups to bind the peptide to the surface through two covalent bonds. The yield of this reaction was quantified through monitoring the absorption of the azide group by GRAS-IR. Despite damage to the monolayer during the reaction, reaction yields of 80−98% were determined for optimized reaction conditions. Although the peptide retains its α-helical configuration under the reaction conditions, GRAS-IR analysis of the amide I and II modes of the surface-bound peptide showed that it is probably randomly oriented on the surface.

Electrostatic fields at the interface of the GTPase H-Ras (Ras) docked with the Ras binding domain of the protein Ral guanine nucleoside dissociation stimulator (Ral) were measured with vibrational Stark effect (VSE) spectroscopy. Nine residues on the surface of Ras that participate in the protein–protein interface were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe into the protein–protein interface. The absorption energy of the nitrile was measured both on the surface of Ras in its monomeric state, then after incubation with the Ras binding domain of Ral to form the docked complex. Boltzmann-weighted structural snapshots of the nitrile-labeled Ras protein were generated both in monomeric and docked configurations from molecular dynamics simulations using enhanced sampling of the cyanocysteine side chain's χ2 dihedral angle. These snapshots were used to determine that on average, most of the nitrile probes were aligned along the Ras surface, parallel to the Ras–Ral interface. The average solvent-accessible surface areas (SASA) of the cyanocysteine side chain were found to be <60 Å2 for all measured residues, and was not significantly different whether the nitrile was on the surface of the Ras monomer or immersed in the docked complex. Changes in the absorption energy of the nitrile probe at nine positions along the Ras–Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and found a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface. These data are used to rationalize several puzzling features of the Ras–Ral interface.

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras–Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras–Raf and Ras–RalGDS complexes.