We describe the development of vibrational spectroscopy as a probe of
electrostatic driving forces in the formation of stable and functional
protein-protein interfaces.  Biological function arises from complex
interactions between cellular macromolecules, such as the organization
of two or more proteins into a functioning assembly.  In the
post-genomic era, enhanced understanding of the cooperativity between
biological molecules is necessary to explore the complexity of living
cells.  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 in this
area, much less is understood about electrostatic influences.  We are
approaching this problem using vibrational Stark effect (VSE)
spectroscopy, in which spectral transitions of molecular probes that are
placed strategically at a protein-protein interface can be related
directly to the strength and direction of electrostatic fields in the
immediate vicinity of the probe.  Here, we present a vibrational
spectroscopic investigation of the electrostatic driving forces that
regulate the formation of a stable interface between the oncoprotein Ras
and a downstream effector RalGDS.  The experimental electrostatic map of
this interface obtained through VSE spectroscopy is connected to kinetic
and thermodynamic studies of Ras-effector binding, and to preliminary
investigations of the interface formed by mutants of Ras that are known
to lead to oncogenic function.