Significancy

The Ras superfamily of small GTPases is responsible for the control of many important cellular processes. These proteins are switched into an active signaling state (“on” state) by GDP to GTP exchange, and return into an inactive “off” state by a GTPase reaction (Figure 1). The defined control of the GTP hydrolysis is of vital importance for the regulation of several signal transduction processes in the living cell. Of high importance for medicinal chemistry here is the major role of the small GTPase Ras in the cell growth-signaling pathway: Site-specific oncogenic mutations inhibit the GTPase reaction, which causes the loss of down regulation of the downstream growth signal. This contributes to uncontrolled cell growth, and results finally in the development of tumors. Common to all members of the Ras superfamily is the catalysis triphosphate hydrolysis by the G-domain: Compared to hydrolysis of GTP in water, the hydrolysis in Ras is accelerated by five orders of magnitude from 200 days to 30 min under physiological conditions. However, to control growth signals in the living cell, GTPase-activating proteins (GAPs,) accelerate catalysis by further five orders of magnitude from 30 minutes to 50 ms.

Main Goals

One of our major research topics is to elucidate in detail, how this remarkable catalysis is obtained. A detailed understanding of structure, dynamics and function of Ras proteins and their oncogenic mutants enables the targeted development of drugs that attack dysfunctional proteins in order to cure diseases. We want to decoded how small GTPases enzymatically catalyzes the GTP hydrolysis and find common catalysis motives. Therefore, we unraveled the structures of the important steps of the catalysis reaction mechanism with sub Ångström resolution.

The enzyme catalysis is mediated by sub-Ångström structural changes of the substrate induced by the protein environment. We have developed and established a method to decode the three dimensional structural information and structural details of spectroscopic data by a combination of FTIR measurements and biomolecular simulations. The time resolved FTIR difference spectroscopy is highly sensitive regarding changes in structure and charge distribution. In combination with biomolecular simulation, we can generate validated structural models with an accuracy of up to 0.01 Å. These structures are experimentally validated by the comparison of the calculated spectral features like vibrational modes, isotopic shifts, and difference spectra with experimental ones. Only this high resolution allows for a complete understanding of catalysis.

The long-term goal of the research field of small GTPases is to understand the effect of oncogenic mutations. Despite of detailed structural insights of ground state of the wild type and mutants the function of oncogenic mutations still remains elusive. It is believed that the mutants effect the transition states or the positioning of the nucleophilically attacking waters. These very rare and transient structures are hard to obtain experimentally and within biomolecular simulations. Therefore, we developed a novel method called Local Mode Analysis (LMA) for calculating IR spectra and assigning spectral IR-bands on the basis of movements of nuclei and partial charges from just a single QM/MM trajectory (Figure 5). Through LMA the decoding of IR spectra no longer requires several simulations or optimizations. The novel approach correlates the motions of atoms of a single simulation with the corresponding IR bands and provides direct access to the structural information encoded in IR spectra.

Beside the investigation on an atomistic level, is it essential to consider the cellular environment on a molecular level as well. Attenuated Total Reflectance (ATR)-FTIR difference spectroscopy provides a tool to analyze membrane bound proteins in a near-native environment. The structure and orientation of the proteins bound to the membrane are decoded from the measured order parameter by bimolecular simulations.