This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-102: Chemist/biologist collaborations facilitating tool development. The objective of the application is to develop a novel tool to study the role of fast protein dynamics in biomolecular recognition based on two- dimensional coherent UV/vis (2DUV/vis) spectroscopy, the theoretical tools to interpret the data, and a biological system with which to develop and validate the technique. Molecular recognition is central to the functions a protein may naturally possess, or those it may be evolved to possess. However, because biomolecular recognition does not generally follow a simple lock-and-key model, it cannot be characterized by structure determination alone. Biological systems generally show a level of induced fit- or conformational selection-like molecular recognition, and thus fluctuations between different protein conformations (i.e. protein dynamics) are critical. Consequently, understanding protein dynamics and how it might be tailored during evolution is fundamental to our understanding of biology. However, unlike technologies to characterize protein structures and relatively slow dynamics, methodologies to identify and characterize fast (pico- to nanosecond) protein motions remain underdeveloped. 2D UV/vis spectroscopy, a state-of-the-art ultrafast laser spectroscopy technique that only very recently has become feasible due to the advent of femtosecond broadband laser sources and phase-sensitive detection techniques for UV/vis signals, provides a multidimensional view of the structure and dynamical fluctuations of a protein. In this application we propose to develop the experimental and theoretical tools required to study protein dynamics using 2DUV/vis spectroscopy. In addition, we propose to develop and validate the experimental and theoretical tools through application to a biological system, evolutionarily related antibody-chromophre complexes, where the important fast motions may be identified and characterized, and for which fast protein dynamics have been predicted to contribute to molecular recognition. As observed during the early development of NMR methods, the application of the proposed 2DUV/vis methods to an important experimental problem should stimulate broader interest and further development, eventually delivering a new and important tool to the scientific community.
The specific aims of this application are:
Specific Aim 1 : Develop 2DUV/vis system capable of experimentally characterizing fast protein dynamics.
Specific Aim 2 : Develop theoretical methodology to interpret the data and design new experiments.
Specific Aim 3 : Explore and validate the application of the developed methodologies, along with X-ray crystallography, to the characterization of biological molecular recognition. The development of methodologies to characterize fast protein dynamics should facilitate the study of many proteins that bind natural chromophores. In addition, there is an increasing number of techniques available to incorporate chromophores into proteins, and the proposed technology development will enable their characterization as well. The proposed research will also have important implications for human health because it will elucidate the mechanisms underlying the evolution of antibodies, our primary line of defense against microbial pathogens and cancer. In addition, great effort has recently been devoted to developing antibody-based therapies, and the results of the proposed should help design rational strategies that optimize the therapeutic potential of antibodies. To successfully address this research challenge, expertise in biology, ultrafast laser spectroscopy, physical chemistry, and theory is required, which is provided through a interdisciplinary collaboration between Drs. Mukamel (physical chemistry, nonlinear spectroscopy, and theory, UC Irvine) and Romesberg (immunology and ultrafast laser spectroscopy, The Scripps Research Institute), thus bridging the disconnect between theory, biophysics, and biology that often limits efforts to apply rigorous physical techniques to biologically relevant problems. The Mukamel lab will perform ab initio simulations of the electronic excitations, compute the 2D signals, interpret the signals in terms of protein structure and dynamics, and design novel pulse sequences to be tested experimentally. The Romesberg lab will implement a 2D UV/vis setup capable of measuring 2D electronic correlation spectra in the visible and near UV spectral region, and perform 2D UV/vis experiments on antibody-chromophore complexes of several sets of evolutionary related antibodies. The feedback between experimental and theoretical methodology development and immediate application to a biologically relevant system will significantly accelerate the refinement of 2D UV/vis spectroscopy as a tool to study biomolecular recognition.
Motions on all timescales contribute to biomolecular recognition, but in contrast to relatively slow dynamics, which may be characterized by a variety of well developed techniques, there is a technology gap for characterizing relatively fast dynamics, and thus, their potential contributions remain poorly understood. We propose to develop 2- dimensional UV/vis spectroscopy to study these fast protein motions, the theoretical tools to understand the data, and a biological system, the evolution of antibodies, to evaluate their importance. In addition to facilitating the study of many other protein- chromophore systems, the proposed research will help to understand how antibodies evolve to protect their host from infection and cancer, and help to devise novel approaches to antibody-based drug development.
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