Warren F. Beck - US grants

Beck, Warren F.
MCB-0091210

Photosynthesis is responsible for most of the biological energy and oxygen on this planet. The first event in photosynthesis is primary charge separation at the reaction center chlorophyll pair. This research program will employ femtosecond coherent Raman spectroscopy to study the excited-state vibrational motions of bacteriochlorophyll dimers. The experimental focus is on the pair of bacteriochlorophylls in the B820 subunit of the purple bacterial light-harvesting protein LH1. B820 shares many of the structural features of the primary electron donor P of the purple bacterial reaction center. The research plan is designed to test the hypothesis that bacteriochlorophyll dimers undergo a nearly barrierless adiabatic surface crossing to a charge-transfer state following excitation of the lower exciton state. The main idea is that certain normal modes of vibration of the bacteriochlorophyll monomer and possibly collective modes of the bacteriochlorophyll dimer serve to mix electronic charge-transfer states with the neutral dimer exciton states. Femtosecond coherent Raman spectroscopy will be conducted with impulsive pump-probe and transient grating methods to detect the vibrational modes that mix the neutral and charge-transfer excited states of the bacteriochlorophyll dimer. The Raman spectra that are obtained with the B820 system will be compared with those that are observed in B777, the monomeric species that is prepared by splitting B820 into single bacteriochlorophyll-polypeptide complexes. In addition, synthetic alpha and beta polypeptides will be used to prepare pure systems containing alpha, beta and beta2 hosts for bacteriochlorophyll dimers. The research program will address how photosynthesis involves paired chlorophyll structures. In broader terms the work will address the role of vibrational coherence in fast chemical reactions in proteins.

The objective of this project, jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and the Experimental Physical Chemistry Program in the Chemistry Division, is to understand the structural nature of the low-frequency normal modes of vibration that control the rate of electron-transfer reactions in proteins. Low-frequency vibrational modes in proteins are of significance to the dynamics of a variety of biological processes, including those of the first events of vision and photosynthesis, because these modes account for the rate-limiting structural rearrangement of the reactant molecules that defines the reaction coordinate. Despite their importance, very little is concretely known about which structures are moving nor is the character of the motion involved in the normal mode of vibration understood. Recent work in the PI's laboratory suggests that intermolecular modes between electronic chromophores and the surrounding protein or solvent are good candidates for the modes that control electron-transfer reactions in proteins. Interactions like these allow a protein to tune the electronic properties and reactivity of the imbedded chromophores. These modes can be detected using a femtosecond time-domain form of resonance Raman spectroscopy called dynamic absorption spectroscopy. This project takes advantage of recent progress made in this laboratory in the detection and analysis of signals arising from low-frequency coherent wave-packet motion on the ground and excited-state potential-energy surfaces of electronic chromophores in solution and in protein hosts. An important finding from this work is that clustered solvent molecules in the first solvation shell of chromophores with delocalized pi-electron systems can be vibronically coupled to the chromophore's pi-to-pi* electronic transition; they obtain resonance Raman activity by attacking the pi-electron density of the chromophore. The initial focus of the research will be on characterization of vibrational coherence of Zn(II)-substituted porphyrins in solution and in two simple protein hosts, cytochrome c and myoglobin. The focus in these studies will be on the ordered polar and non-polar intermolecular interactions of neighboring solvent or amino acid side chains with the pi-electron density of the porphyrin.

The methods and ideas that will be developed in this project are potentially of broad use in the study of chemical dynamics in biological systems, materials, and in condensed phases. The use of strong electronic chromophores in time-resolved spectroscopic studies of protein and solvent dynamics is also of potential importance to the area of protein stability and folding/misfolding. This research will play an integral role in the PI's graduate and undergraduate teaching by providing key lecture topics and applications even in introductory courses. Additionally, the work will provide excellent training for research students at all levels. The work will expose the students to a wide range of disciplines, ranging from structural biology to chemical physics.

This project employs time-resolved fluorescence spectroscopy to study how small proteins change in structure when they are displaced from the equilibrium or native structure. The planned research exploits a new method developed in this laboratory that uses the vibrational energy transferred from an excited-state electronic chromophore to drive the host protein to which it is attached from its native structure to a range of partially unfolded states that probably correspond to late intermediates along the folding/unfolding pathway. Because the intermediate structures are generated optically under the solution conditions that favor the native structure, the refolding reactions that follow the production of unfolded intermediates can be characterized with independent control of the solvent composition or temperature over a very wide range of timescales, over the picosecond to millisecond regime, through the use of prompt and delayed fluorescence methods. Zinc(II)-substituted and metal-free cytochromes c are the main systems to be studied. The porphyrin chromophore in cytochrome c serves in the planned experiments as both the trigger and the sensor for the unfolding and refolding reactions. A tryptophan residue will also be used as a remote sensor for the reactions that are triggered by the porphyrin. This research provides a direct test of the central hypothesis that proteins fold rapidly under physiological conditions because they move randomly on a funnel-shaped potential-energy surface that has the native structure at the funnel's minimum. The research will also have an impact on the broader fields of protein structure and function that relate to the barrier-crossing processes that lead to conformational changes during photobiological and enzyme-catalyzed reactions.

This project plays an integral part in the teaching of undergraduate and graduate courses by the PI and co-PI. It provides key lecture topics and applications even in introductory courses. Research participants are exposed to a wide range of disciplines ranging from structural biology to chemical physics. Undergraduate students are encouraged to start working in the laboratory in the freshman year, and they usually contribute to publications by the senior year. Additionally, this project is associated with a new outreach program that will feature visits by the PI and co-PI to high schools in the Lansing, Michigan area to give presentations to Chemistry and Physics classes on the use of laser spectroscopy in biophysical chemistry, structural biology, and related disciplines. Interested students and their teachers will then be invited to visit the PI, co-PI, and their graduate students, to see the research laboratories, to talk about careers in science and engineering, and perhaps to get involved in research projects. This outreach plan will lead to interactions with students from underrepresented groups who will be recruited to undertake undergraduate studies in the sciences at Michigan State University. This effort is intended to augment an existing effort by the Department of Chemistry at MSU to recruit and retain students from underrepresented groups.

Abstract
This proposal is for the construction of an instrument that will provide previously unobtainable information on the organization and dynamics of plasma membranes, biomimetic structures, and light harvesting proteins. The dissipation of energy within lipid bilayers and light harvesting proteins is not well understood but is thought to mediate the function of these two different classes of biomolecular systems. One of the broad biological issues this project addresses is the fundamental, molecular basis for the formation of lipid raft structures, and why such structures vary with the concentrations and identities of the bilayer constituents. Understanding the molecular structural basis for lipid raft organization requires that the viscoelastic properties and intermolecular interactions of the lipid bilayer constituents can be measured. The proposed instrumentation will allow for the measurement of molecular motion and thermal energy flow in biomolecular systems.
This project entails designing, constructing, and characterizing an instrument that will apply stimulated-emission spectroscopy to the study of structure and dynamics of biological membranes and proteins. The instrument will reveal how the lipid and protein components of the mammalian cell membrane interact to yield functional assemblies that are responsible for energy transduction, transmembrane transport, and molecular recognition. The instrument will characterize the lipid-lipid and lipid-protein interactions that control the fluidity of the lipid bilayer assembly and the flow of thermal energy between components that serves as the driving force for chemical reactions and molecular motion.
The instrument will employ tunable picosecond lasers in a two-color pump?probe detection scheme to examine vibrational energy-transfer and fast molecular-scale motions in bilayer membranes and light harvesting proteins. Such measurements have not been possible before. The detection system is phase-sensitive and shot-noise-limited to measure transmission changes of one part in 107. Because the reaction dynamics underlying the formation and decay of short-lived complexes in membranes are controlled by vibrationally activated barrier-crossing processes, the information obtained with the proposed instrument is crucial to reaching an understanding of the dynamics associated with spatially heterogeneous structures, including lipid rafts and proteins.
The creation of broadly accessible instrumentation that advances the state of the art in the measurement of lipid bilayer properties and dynamics will have a major impact on the MSU and regional scientific communities as well as on the global scientific community. The PI and co-PIs collaborate with faculty in a host of other MSU departments (e.g. Biochemistry and Molecular Biology, Food Safety and Toxicology, Cell and Molecular Biology, Microbiology and Molecular Genetics), faculty from nearby institutions (e.g. Saginaw Valley State University, Western Michigan University) and from international institutions (e.g. University of Warsaw (Poland), University of Bath (UK), National University of Singapore, and Shaanxi Normal University (PRC)). They also collaborate with Federal research organizations such as the US Army Engineer Research and Development Center in Champaign, IL.
Broadening inclusion of under-represented groups in science is critically important. Michigan State University has multiple programs in place to connect with under-represented groups at the high school (MSU High School Honors Science/Math/Engineering Program (HSHSP), ACS Project SEED), undergraduate (National Organization of Black Chemists and Chemical Engineers (NOBCChE), DREW/TAC Program), graduate (NOBCChE, MSU African-American, Latino(a)/Chicano(a), Asian/Pacific American, and Native American (ALANA) Program) and post-graduate levels (MSU sponsored minority post-doctoral fellowships). The PI and the co-PIs have collaborated with these programs on several levels and continue to strive to provide students from all groups with hands-on research opportunities and mentoring.

Carotenoids are essential light-absorbing pigments that help conversion of sunlight energy during photosynthesis and, in addition, provide photoprotection from the sun's damaging effects to photosynthetic organisms. With this award, the Chemistry of Life Processes Program in the Chemistry Division and the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences is funding Dr. Gascon from University of Connecticut and Dr. Beck from Michigan State University to investigate the energy transfer and photoprotection functions of carotenoids in the proteins of such organisms. High-level computational methods are developed to predict how light harvesting affects the structure of carotenoids within these proteins and experimental methods are used to test these predictions. Results from these studies are applicable to engineering new materials for solar energy capture and storage that are more efficient at absorbing light, while being less susceptible to photodamage. In addition, the project increases the knowledge of high school and undergraduate students, including those from underrepresented groups, on how computational methods help to answer important questions in biology and chemistry, and how computer modeling is prevalent and impacts our daily lives.

Carotenoids act as light-harvesting pigments, which are uniquely positioned to absorb light outside the light-capturing range of Chlorophylls (Chl). Equally important is the role of carotenoids as photo-protectors of the photosynthetic apparatus via non-photochemical quenching mechanisms and quenching of singlet oxygen. Two examples of protein complexes with these two capabilities are the peridinin-chlorophyll a-protein (PCP) from marine algae dinoflagellates and the orange carotenoid protein (OCP) from cyanobacteria. Despite the availability of high-resolution structures of PCP, OCP, and other carotenoid-containing light-harvesting proteins, and considerable experimental and theoretical efforts, a general understanding of how the carotenoids function in energy transfer and photoprotection is still not fully available due to longstanding controversies in several areas. This research produces a systematic and synergistic examination of these mechanisms, combining advanced electronic structure calculations in proteins and advanced spectroscopy techniques to discover the controlling spectral factors and the dynamical fate of carotenoids during the first events of light harvesting and photoprotection in photosynthetic proteins.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

With the support of the Chemistry of Life Processes Program in the Chemistry Division, Professor José Gascón from University of Connecticut (UConn) and Professor Warren F. Beck from Michigan State University will investigate the mechanisms that cyanobacteria employ to protect themselves from damage due to excessive exposure to sunlight. This project will determine how a ketocarotenoid bound to the orange carotenoid protein (OCP) serves as a sensor for the light intensity and color. The main goal is to determine the photophysical mechanism that converts the OCP from a resting, orange form (OCPO) to an active, red form (OCPR); this conversion protects the organism during high intensity solar illumination by slowing the rate of photosynthetic energy storage. The work will provide insight in how the components in the photosynthetic apparatus of cyanobacteria and green plants work together to store solar energy in molecular form and to avoid light-driven side reactions that cause damage. The new knowledge could impact the design and engineering of materials that can be used in solar energy applications. The research will afford an excellent opportunity for graduate students involved to learn advanced computer modeling and spectroscopy techniques and their application to advanced studies of molecular structure and function. In addition, the project will create research opportunities for high school students from the School of Exploratory Chemical Research and Training (SECRET) and undergraduate students who participate in the NSF-funded Research Experiences for Undergraduates (REU) site in Chemistry at UConn.<br/><br/>The planned research involves a combination of state-of-the-art femtosecond multidimensional electronic spectroscopy and ultrasensitive fluorescence methods to sense the conformational changes that the ketocarotenoid undergoes in OCPO when it absorbs blue-green light. Electronic structure calculations and molecular dynamics simulations will then be used to develop a molecular model for the first structural events that follow photoexcitation of the ketocarotenoid. The research plan will test the hypothesis that light-driven conformational changes of the ketocarotenoid initiate the structural reorganization of the protein. These studies are expected to provide molecular details of how the ketocarotenoid senses ambient solar illumination.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.