Collaborative Research Dissecting photoconversion in fluorescent proteins frame by frame

Project: Research project

Description

Senior Personnel: PI: Wachter, Rebekka M. (Arizona State University, School of Molecular Sciences). Co-PI: Fang, Chong (Oregon State University, Department of Chemistry). Collaborator: Mehl, Ryan A. (Oregon State University, Department of Biochemistry and Biophysics). Collaborator: Campbell, Robert E. (University of Alberta, Department of Chemistry). Collaborator: Lin, Su (Arizona State University).

Overview. The overarching goal of this work is to investigate how ultrafast atomic motions in a proteins active site are linked to protein dynamics and catalytic functionality. This question will be studied using green-to-red photoconvertible fluorescent proteins (pcFPs) as a model system. pcFPs constitute a large group of green fluorescent proteins (GFPs) known to undergo an irreversible color conversion process that has been exploited extensively in super-resolution imaging applications. Recently, evidence has been provided that the natural evolution of pcFPs is linked to changes in protein motions that involve the relocation of a knob-like anchoring region diagonally across the beta barrel fold. In work proposed here, dynamic motions will be probed on a variety of time scales, using femtosecond stimulated Raman spectroscopy (FSRS) in combination with photoconversion kinetic measurements and molecular dynamics simulations. This work will involve the incorporation of isotopically labeled and non-canonical amino acids. The synergistic experimental and modeling approach will allow for the correlation of dynamical and chemical processes over a broad range of timescales.

Intellectual Merit. To develop a mechanistic model that links protein motions to key chemical events, a series of pcFPs will be tested that exhibit varying degrees of photoconversion efficiency. First, the role of ultrafast dynamics in facilitating chemical steps will be examined using wavelength-tunable FSRS, currently the only table-top technique that can track bond vibrations starting with photoexcitation. This approach will allow for the characterization of primary events in light-induced chromophore deformations that prepare the active site for the chemical steps that yield an expanded chromophore pi-system.

Second, molecular dynamics simulations will be carried out to model different structural intermediate states. Such states will be examined further by electrostatics calculations, to address whether transient active site repacking produces charge redistributions predictive of photoconversion efficiency. Third, various mechanistic hypotheses involving electrostatic and dynamic features will be tested by performing photoconversion measurements. Kinetic and equilibrium constants will be determined with and without the incorporation of non-canonical amino acid residues. Kinetic isotope effect studies will be employed to identify rate-limiting events. With this synergistic approach, functional models will be developed that integrate ultrafast motions in the active site, local side chain rotations and global protein chain dynamics on the pathway of green-to-red photoconversion.

Broader Impacts. The proposed research will serve as a cornerstone for several outreach activities, in particular educational High School visitation programs. At ASU, the PI (Wachter) is involved in the SCience and ENgineering Experience (SCENE) program, and will host one or two high school students per year in her laboratory. This effort will provide cutting-edge research experience to high school students over 16 years old, thereby encouraging them to choose a career in the sciences. At OSU, the PI (Fang) recently secured an Action Research Fellowship through ESTEME@OSU funded by NSFs WIDER program (DUE 1347817). He intends to research and implement evidence-based instructional practices to enhance physical chemistry education at OSU. Both PIs will integrate cutting-edge research in biophysics and molecular biology into formal coursework, such as a large (300-student) undergraduate physical chemistry course, as well as a smaller senior elective course in protein biochemistry.

Keywords: Hands-on Training of High School Students; High School Visitation Weekends; Integration of Research and Teaching; Evidence-based Instruction; Dissemination of Research Results to the General Public.
StatusActive
Effective start/end date7/1/186/30/21

Funding

  • National Science Foundation (NSF): $550,000.00

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Proteins
Biophysics
Students
Physical chemistry
Biochemistry
Chromophores
Kinetics
Raman spectroscopy
Molecular dynamics
Electrostatics
Knobs
Amino Acids
Molecular biology
Relocation
Photoexcitation
Equilibrium constants
Computer simulation
Green Fluorescent Proteins
Isotopes
Teaching