R01AI175067

"On the fly" time resolved cryo-EM studies of intermediate HIV-1 RT transition states - Summary

Historically, interactions between substrates and/or inhibitors and enzymes have been viewed in terms of binding of small molecules to relatively rigid protein targets. However, computational and experimental studies have revealed that many proteins, in particular DNA polymerases, undergo molecular motions over a wide range of timescales. Such conformational flexibility is critical for enzymatic activity, drug action, and drug resistance.

Moreover, contemporary structural biology approaches, such as X-ray crystallography, only have the ability to resolve the structures of thermodynamically stable species and cannot inform on kinetic intermediates. Our group has developed cutting-edge technology - "on-the-fly" time-resolved cryo-electron microscopy (EM) - that, for the first time, facilitates visualization of novel protein conformations, including those transiently occurring during catalysis.

Specifically, we have developed two TR cryo-EM approaches: (I) rapid chemical mixing of enzyme and substrates; and (II) photo-activation that takes advantage of caged substrates and/or amino acid residues. We have developed substantial preliminary data that support the feasibility and power of these approaches.

In this application, we will apply this technology to address biologically and clinically relevant knowledge gaps in HIV-1 reverse transcriptase (RT) biology, with specific focus on the incorporation of nucleosides and nucleoside inhibitors, nucleoside inhibitor resistance, and the relationship between DNA synthesis and ribonuclease H (RNASE H) activity.

University Of Pittsburgh - Of The Commonwealth System Of Higher Education

TO ASSIST PUBLIC AND PRIVATE NONPROFIT INSTITUTIONS AND INDIVIDUALS TO ESTABLISH, EXPAND AND IMPROVE BIOMEDICAL RESEARCH AND RESEARCH TRAINING IN INFECTIOUS DISEASES AND RELATED AREAS, TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS. TO ASSIST PUBLIC, PRIVATE AND COMMERCIAL INSTITUTIONS TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS, TO PROVIDE RESEARCH SERVICES AS REQUIRED BY THE AGENCY FOR PROGRAMS IN INFECTIOUS DISEASES, AND CONTROLLING DISEASE CAUSED BY INFECTIOUS OR PARASITIC AGENTS, ALLERGIC AND IMMUNOLOGIC DISEASES AND RELATED AREAS. PROJECTS RANGE FROM STUDIES OF MICROBIAL PHYSIOLOGY AND ANTIGENIC STRUCTURE TO COLLABORATIVE TRIALS OF EXPERIMENTAL DRUGS AND VACCINES, MECHANISMS OF RESISTANCE TO ANTIBIOTICS AS WELL AS RESEARCH DEALING WITH EPIDEMIOLOGICAL OBSERVATIONS IN HOSPITALIZED PATIENTS OR COMMUNITY POPULATIONS AND PROGRESS IN ALLERGIC AND IMMUNOLOGIC DISEASES. BECAUSE OF THIS DUAL FOCUS, THE PROGRAM ENCOMPASSES BOTH BASIC RESEARCH AND CLINICAL RESEARCH. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM EXPANDS AND IMPROVES PRIVATE SECTOR PARTICIPATION IN BIOMEDICAL RESEARCH. THE SBIR PROGRAM INTENDS TO INCREASE AND FACILITATE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM STIMULATES AND FOSTERS SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. RESEARCH CAREER DEVELOPMENT AWARDS SUPPORT THE DEVELOPMENT OF SCIENTISTS DURING THE FORMATIVE STAGES OF THEIR CAREERS. INDIVIDUAL NATIONAL RESEARCH SERVICE AWARDS (NRSAS) ARE MADE DIRECTLY TO APPROVE APPLICANTS FOR RESEARCH TRAINING IN SPECIFIED BIOMEDICAL SHORTAGE AREAS. IN ADDITION, INSTITUTIONAL NATIONAL RESEARCH SERVICE AWARDS ARE MADE TO ENABLE INSTITUTIONS TO SELECT AND MAKE AWARDS TO INDIVIDUALS TO RECEIVE TRAINING UNDER THE AEGIS OF THEIR INSTITUTIONAL PROGRAM.

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

Pittsburgh, Pennsylvania 152221808 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 312% from $741,636 to $3,057,315.