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Electrostatic Fields at the Protein-Protein Interface

Summary: Macromolecular interactions in biological systems are now a major focus of interest.  In the post-genomic era, enhanced understanding of the cooperation between biological molecules such as proteins, DNA, RNA, and lipids is necessary to explore the complexity of living cells.  Macromolecular interactions lead to emergent properties necessary for life, but can only be studied or understood if the molecular-level details that drive and control those interactions are themselves understood.  Furthermore, molecules that promote or disrupt specific macromolecular interactions have vast pharmacological potential.  The affinity and specificity of macromolecular interactions are the result of both structural and electrostatic driving forces, but while the field of structural biology has made great advances, much less is understood about electrostatic influences.  The Webb group measures electrostatic fields at protein-protien intrafaces and seeks to develop computational models that accurately predict these interactions.  We do this using vibrational Stark effect (VSE) spectroscopy, in which spectral shifts of a probe oscillator's energy is related directly to that probe's local electrostatic environment.

Electrostatic fields in proteins: The highly organized three dimensional structure of a protein can support large internal electrostatic fields that influence every aspect of the protein's function including folding, chemical reactivity and kinetics, and protein-protein interactions.  Until recently, there was no direct experimental method for accurately measuring such fields in a protein.  Much theoretical work has focused on calculating electrostatic fields in a protein of known structure, but in very few cases have these predictions been compared to or verified by experimental studies.  A new approach to measuring electrostatic fields uses the vibrational Stark effect, the shift in absorption energy of a molecular vibration caused an electrostatic field and measured by Fourier transform infrared spectroscopy (FTIR).  

Vibrational Stark effect (VSE) spectroscopy:  The principle of VSE spectroscopy is summarized in the figure below:



When a molecule, for example the nitrile functional group, is placed in an applied external electric field (F(ext)) and illuminated, absorption features will be shifted by an amount deterined by the strength and direction of the local dipole moment change (delta mu) asociated wth that transition.  This phenomenon is a general statement of the Stark effect.  When delta mu is aligned perpendicular to the field the transition will not be affected, but dipoles aligned parallel or antiparallel to the applied field will have transitions shifted to lower and higher energy, respectively.  With a sample of immobile randomly oriented molecules, the application of a field leads to a general broadening of the absorption spectrum.  The magnitude of delta mu, also called the Stark tuning rate, is obtained from the field-on minus field-off dufference spectrum (delta A).

While this approach can be applied to any type of transition, molecular vibrations are particularly attractive as probes since they are localized and directional (delta mu lies parallel to the bond axis).  Once the vibration's Stark tuning rate has been calibrated, the probe is placed into a protein using methods described below.  In this part of the measurement, no external field is applied; instead, the probe is exposed to the electrostatic field supported by the protein, which shifts the vibrational absorption frequency of the probe.  Shifts in the probe's absorption frequency are related to the change of the protein electrostatic field (delta F(protein)) through the field equation shown in the figure.

Putting probes into proteins:  An ideal VSE probe must have a vibrational absorption well removed from the protein / buffer background, and therefore must be an unnatural functional group.  The nitrile group has proven to be an exceptionally useful probe and is the current focus of our work.  The first step of our experiment therefore involves inserting a single nitrile group into a specific, known location inside a protein.  We use the tools that are now common throughout the molecular biology community for inserting unnatural functional groups into proteins.  There are four techniques that we use interchangably depending on the experiment and the protein being studied:

               1) posttranslational modification of natural amino acid residues
               2) 
binding a ligand or cofactor carrying the functional group to the protein
               3) semisynthetic incorporation of an unnatural amino acid carrying the functional group into a small peptide
               4) biosynthetic incorporation of an unnatural amino acid carrying the functional group

Because our probe is a diatomic molecule, the perturbation to the protein is minimal.  Verifying that this is true by fully characterizing protein structure and funtion whenever a new probe is inserted into the protein is an important part of our experiment.

People:  Elisa Novelli, Josh Slocum, Ben Chang, Desiree Fernadez

Publications: 
Ensign, D.L. and Webb, L. J.  "Factors Determining Electrostatic Fields at the Ras/Effector Interface."  Proteins 2011, 79, 3511-3524.  pdf

Stafford, A. J.; Walker, D.; Webb, L. J.  Electrostatic Effects of Mutations to Ras Glutamine 61 Measured Using Vibrational Spectroscopy of a Thiocyanate Probe."  Biochemistry 2012, 51, 2757-2767. pdf

Ragain, C. M.; Newberry, R. W.; Ritchie, A. W.; Webb, L. J.  "The Role of Electrostatics in Differential Binding of RalGDS to Rap Mutations E30D and K31E Investigated by Vibrational Spectroscopy of Thiocyanate Probes."  J. Phys. Chem. B 2012, 116, 9326-9336pdf

Walker, D. M.; Wang, R.; and Webb, L. J.  "Conserved Electrostatic Fields at the Ras-Effector Interface Measured Through Vibrational Stark Effect Spectroscopy Explain the Difference in Tilt Angle in the Ras Binding Domain of Raf and RalGDS."  Phys. Chem. Chem. Phys.  2014, 16. 20047-20060.  pdf

Ritchie, A. R.; Webb, L. J.  "Understanding and Manipulating Electrostatic Fields at the Protein-Protein Interface Using Vibrational Spectroscopy and Continuum Electrostatics Calculations."  J. Phys. Chem B 2015, 119, 13945-13957. pdf


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