Molecular alignment of denatured states of staphylococcal nuclease with strained polyacrylamide gels and surfactant liquid crystalline phases.
Residual dipolar couplings reflect the orientation of vectors between pairs of magnetic nuclei relative to a unique set of molecular axes. Thus, unlike NOEs and scalar couplings, dipolar couplings provide access to long-range structural information. A prerequisite for measurement of these NMR parameters is imposition of a weak net alignment, most simply by forcing the macromolecules to tumble in an asymmetric environment that restricts some orientations more than others. In this report, several denatured forms of staphylococcal nuclease are aligned by using compressed and stretched polyacrylamide gels, a nonionic type of lipid bilayer disk or bicelle, and a liquid crystalline phase formed by a cationic lipid. All three types of media can be used at high urea concentrations. While polyacrylamide gels and bicelles produce similar alignment tensors through steric interactions, a liquid crystalline phase of cetylpyridinium bromide aligns denatured nuclease along a different set of axes, presumably through electrostatic effects. The analysis of residual dipolar couplings collected with two different alignment tensors may permit the calculation of ensembles of conformations. The dipolar couplings observed for staphylococcal nuclease denatured with urea, by low pH or by deletion of residues from both termini, suggest that all denatured forms share a common "topology", one which has been shown previously to be native-like. Although SDS/nuclease complexes give sharp and disperse (1)H-(15)N correlation spectra, only small couplings are observed in strained polyacrylamide gels.
Ackerman
Michael S
MS
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Shortle
David
D
D000143:Acids / Q000737:chemistry; D002594:Cetylpyridinium / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D000441:Hexanols / Q000737:chemistry; D008836:Micrococcal Nuclease / Q000737:chemistry*; D011489:Protein Denaturation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013210:Staphylococcus / Q000201:enzymology*; D014508:Urea / Q000737:chemistry
D000143:Acids; D000441:Hexanols; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D002594:Cetylpyridinium; D008836:Micrococcal Nuclease