Project Grant F32GM161090

INVESTIGATING AND TARGETING CONFORMATIONAL LANDSCAPE IN CLASS I RIBONUCLEOTIDE REDUCTASE - PROJECT SUMMARY/ABSTRACT RIBONUCLEOTIDE REDUCTASE (RNR) IS A CRUCIAL ENZYME IN ALL ORGANISMS RESPONSIBLE FOR SYNTHESIZING ALL FOUR DEOXYRIBONUCLEOTIDES (DNTPS), THEREBY CONTROLLING THE BALANCE OF NUCLEOTIDE POOLS REQUIRED FOR DNA REPLICATION AND REPAIR. CLASS IA RNR, WHICH IS COMMON FOR HUMANS AND E.COLI, REQUIRES TWO SUBUNITS FOR ITS ACTIVITY: THE A2 SUBUNIT CONTAINS THE ACTIVE SITE, AND THE B2 SUBUNIT HOUSES A DIFERRIC TYROSYL RADICAL COFACTOR. NUCLEOTIDE REDUCTION REQUIRES TWO SUBUNITS TO FORM THE ACTIVE COMPLEX A2B2. WITHIN THIS COMPLEX, EACH TURNOVER REQUIRES UNPRECEDENTED LONG-RANGE (~32 A) REVERSIBLE RADICAL TRANSFER (RT) FROM B2 TO A2 VIA A SERIES OF SIX AMINO ACID RADICAL INTERMEDIATES. BECAUSE OF ITS ESSENTIAL ROLE, RNR HAS LONG BEEN RECOGNIZED AS A PROMISING TARGET FOR CANCER THERAPY AND FOR THE DEVELOPMENT OF ANTIBACTERIAL AND ANTIVIRAL AGENTS, LEADING TO THE DEVELOPMENT OF MULTIPLE INHIBITOR CLASSES DESIGNED TO BLOCK ITS ACTIVITY. UNFORTUNATELY, COMPOUNDS TARGETING THE COFACTOR OR SUBSTRATE-BINDING SITE OFTEN EXHIBIT LIMITED SELECTIVITY AND HIGH CYTOTOXICITY. AN INCOMPLETE UNDERSTANDING OF RNR DYNAMICS IN ALL ORGANISMS HAS CONSTRAINED PROGRESS TOWARD NEW THERAPEUTIC CANDIDATES. FOR INSTANCE, RT IN RNR HAS BEEN POSTULATED TO BE CONFORMATIONALLY GATED; HOWEVER, THE PRECISE CONFORMATIONAL CHANGES THAT CONTROL TURNOVER REMAIN POORLY DEFINED. ADDING TO THIS COMPLEXITY, THE MOLECULAR BASIS OF REGULATORY SWITCHING BETWEEN ACTIVE AND INACTIVE OLIGOMERIC STATES (A6 IN HUMANS AND A4B4 IN E. COLI) REMAINS ELUSIVE. ALL OF THESE, TOGETHER WITH THE ABSENCE OF A ROBUST SCREENING STRATEGY, REPRESENT A KEY BARRIER TO THE RATIONAL DESIGN OF NOVEL SELECTIVE RNR MODULATORS. THIS PROPOSAL AIMS TO ADDRESS THESE BARRIERS BY UTILIZING: (1) STOPPED-FLOW FLUORESCENCE MEASUREMENTS TO DEFINE THE DYNAMICS OF C-TERMINAL B-TAIL INSERTION INTO THE A-SUBUNIT AS A CANDIDATE GATING EVENT; (2) NEW BIOPHYSICAL TOOLS TO MONITOR AND CHARACTERIZE INTERCONVERSION BETWEEN THE ACTIVE AND INACTIVE RNR FORMS; (3) DEVELOPMENT OF A HIGH-THROUGHPUT PLATFORM FOR DISCOVERING SMALL, NON-NUCLEOSIDE MOLECULES THAT SELECTIVELY TARGET RNR STATES. ADDRESSING THESE AIMS WILL OVERCOME KEY BARRIERS TO UNDERSTANDING CONFORMATIONAL LANDSCAPE OF RNR AND ESTABLISH A PRACTICAL FRAMEWORK FOR DEVELOPING NOVEL RNR MODULATORS. THE NOCERA LAB AT HARVARD UNIVERSITY PROVIDES AN IDEAL ENVIRONMENT TO ACHIEVE THE PROPOSED RESEARCH AND TRAINING OBJECTIVES, FORMING A STRONG FOUNDATION FOR A FUTURE ACADEMIC CAREER. PROF. NOCERA'S EXPERTISE IN PHOTOCHEMISTRY, SPECTROSCOPY, AND INORGANIC CHEMISTRY, ALONG WITH MENTORSHIP FROM PROF. STUBBE, OFFERS INVALUABLE TRAINING IN ADVANCED ENZYMATIC METHODOLOGIES, INCLUDING UNNATURAL AMINO ACID INCORPORATION, KINETIC AND BIOPHYSICAL TECHNIQUES (E.G., STOPPED-FLOW, EPR, ETC.), AND PERFORMING LASER-INDUCED PHOTOCHEMICAL REACTIONS. ADDITIONALLY, HARVARD PROVIDES EXTENSIVE OPPORTUNITIES FOR DEVELOPING SKILLS IN SCIENTIFIC MENTORSHIP, MAKING IT AN IDEAL ENVIRONMENT TO PURSUE RESEARCH GOALS AND PREPARE FOR A SUCCESSFUL CAREER IN ACADEMIA.