Deuterium incorporation into Escherichia coli proteins. A neutron-scattering study of DNA-dependent RNA polymerase

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Abstract
Neutron small‐angle scattering studies of single protein subunits in a protein‐DNA complex require the adjustment of the neutron scattering‐length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA‐dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small‐angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements.