Large α‐helical movements observed in ternary crystals of RB69 DNA polymerase following nucleotide incorporation

Abstract

Raman difference spectroscopy is used to monitor the conformational changes associated with dNMP incorporation in single crystals of a RB69 DNA polymerase (RB69pol) ternary complex. An inactive crystal of L415G RB69pol exo‐ with a primer/template duplex and a cognate dATP was produced in the presence of Ca2+. The Raman spectrum of the crystallized complex was obtained using a Raman microscope. Phosphodiester bond formation is triggered by soaking in Mg2+. The Raman difference spectra [ie, (ternary complex + Mg2+)–(ternary complex with no Mg2+)] show that conformational changes are complete prior to the acquisition of the first Raman spectrum following magnesium incorporation into the crystal. Polyacrylamide gel electrophoresis experiments carried in parallel show that dNMP incorporation occurs rapidly in the crystal. The Raman data indicate that significant changes in protein amide features, tyrosine ring modes, and nucleic acid base backbone modes have occurred. Overall, they are dominated by the dichroic effect with the molecular groups changing orientation with respect to the laser beam following nucleotide incorporation. The amide I and amide III α‐helix features are unusually narrow and are assigned to movement of the P and N helices. These results demonstrate the ability of Raman spectroscopy to monitor conformational changes in proteins via dichroism. The Raman spectra and the gel electrophoresis support the hypothesis that the major spectroscopic changes are because of structural changes in the ternary complex crystal following correct dNMP incorporation. Copyright © 2015 John Wiley & Sons, Ltd.