This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Conformational switching is an essential feature of the function of nearly all known enzymes. Small angle x-ray scattering (SAXS) is a sensitive technique for detecting size and conformation changes of macromolecules in solution, but it is sparingly used in biochemistry. It is particularly useful for molecules outside the range accessible to NMR (>20 kDa). In this study SAXS will be used to examine the global Conformational states for several DNA polymerases as a function of different solution conditions. Taq polymerase is the predominant polymerase used in PCR (the polymerase chain reaction). There currently exist two rather different crystal structures for Taq polymerase: an elongated conformation and a compact form. Since the two different conformations dramatically change the distance between the two enzymatic active sites of the polymerase, the resolution of which forms exist in solution has significant functional implications. Measurements of the molecular radius of gyration (Rg) as a function of solution conditions in this study will determine if Taq polymerase can occupy either conformation in solution, or if it predominantly occupies only one of the two conformations. The expansion of Taq and 3 other DNA polymerases as a function of thermal and chemical denaturation will also be examined. Information from unfolding experiments performed on these structurally homologous polymerases surprisingly suggests that their denatured states are very different in size. SAXS measurements will directly test this hypothesis. In addition to the polymerase measurements, this study will also measure Rg changes for the enzyme aspartate transcarbamylase (ATCase) as a function of bound ligand. ATCase has been known for many years to switch between two different Conformational states (T and R) during its normal functional cycle. Recent data from several labs suggests the Conformational switch in ATCase may be altered while retaining the allostenc functions associated with the conformational switch.
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