VISUALTRANSPORT · Visualizing Functional Structural Dynamics of Membrane Protein Transporters

Visualizing Functional Structural Dynamics of Membrane Protein Transporters

Proteins rearrange their structures to carry out specific functions. To understand protein function, we therefore need to monitor and display protein structural dynamics, preferably in the natural environment. In this research project, protein structural rearrangements of membrane protein transporters were monitored using a combined computational-experimental approach. The aim was to characterize protein-mediated membrane transport in solution at room temperate, i.e. as close to the natural conditions as possible. The protein targets were membrane protein transporters from the P-type ATPase family with specificity for Ca2+, Cu+, and Zn2+ ions, respectively. Protein targets were either isolated from rabbit (Ca2+ ATPase) or produced using equipment for recombinant membrane protein preparation (Cu+ and Zn2+ ATPases), which was optimized in this project. To achieve strong signal in time-resolved experiments it is critical to activate as many protein molecules as possible to resolve reaction intermediates in the reaction cycle. Infrared spectroscopy was used to optimize triggering of the transport reaction by means of UV-laser induced activation of caged ATP to release ATP and, hence, initiate the ATP-dependent protein reaction. Such time-resolved FTIR experiments have never been performed on a full-length protein from the heavy-metal P-type ATPase transporters. Importantly, the spectroscopy characterization paved way for time-resolved X-ray scattering experiments conducted at the European Synchrotron Radiation Facility (ESRF) in France where X-ray data were collected for the Ca2+, Cu+, and Zn2+ ATPases. The well-characterized Ca2+ transporter was used as proof-of-principle system for the computational structural refinement of the low-resolution experimental data. The combined experimental data and computational refinement allow access to protein transition states that cannot be captured by X-ray crystallography or cryo-EM methods. The developed methodology and recorded structural data are therefore of great interest to the structural biology field. In addition, time-resolved X-ray scattering experiments on a millisecond time scale has previously only been conducted on light-sensitive proteins. The successful experiments were triggered by a caged compound (caged ATP) and therefore pave way for an entirely new range of medically relevant biological targets given an ever-increasing number of caged compounds, such as caged ions and caged neurotransmitters. Because the data generated from scattering experiments are of low-resolution character, a structural refinement tool is crucial to obtain high-resolution structural information. Therefore, a structural refinement tool based on metadynamics simulations was developed. A major advantage of the published approach compared to other refinement techniques is that it enables identification of a unique structural solution by resolving the free energy landscape with respect to the experimental data. Computational methods were also used to extract dynamical information not accessible to experimental efforts. Using atomistic computer simulations, crucial structural dynamics were determined in the Cu+- and Zn2+-transporters. Specifically, the Cu+ ion release pathway and associated energetics were determined using a combined experimental-computational approach. A similar approach was used to characterize the elusive Cu+ entry pathway using molecular modeling, computer simulations and biochemical characterization. In addition, the structural dynamics associated with ion release were determined using the first crystal structures of a Zn2+ transporting P-type ATPase. The structural features associated with reaction cycle dynamics determined in this project provide important clues to understand the fundamental physic-chemical properties underlying protein function. This can potentially have significant impact on human health. First, atomistic structural features of Cu+ transport are critical to understand hereditary disease (Menkes and Wilsons diseases). In addition, because the Zn2+ ATPase ZntA is lacking in human, but is prevalent in pathogens, it is an attractive target for developing novel antibiotics to fight multi-drug resistance.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз