Solution NMR of proteins within polyacrylamide gels: diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes.
The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-15N T2 (T1) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to deriveresidual dipolar couplings for human HIV-1 Nef and ubiquitin.
Sass
H J
HJ
Department of Structural Biology, Biozentrum, University of Basel, Switzerland.
Musco
G
G
Stahl
S J
SJ
Wingfield
P T
PT
Grzesiek
S
S
D000180:Acrylic Resins / Q000737:chemistry* / Q000494:pharmacology; D016880:Anisotropy; D004058:Diffusion / Q000187:drug effects; D015693:Gene Products, nef / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry; D006801:Humans; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D018717:Purple Membrane; D012191:Retroviridae Proteins / Q000737:chemistry; D012996:Solutions; D014452:Ubiquitins / Q000737:chemistry; D014867:Water / Q000494:pharmacology; D054311:nef Gene Products, Human Immunodeficiency Virus
D000180:Acrylic Resins; D015693:Gene Products, nef; D009587:Nitrogen Isotopes; D011506:Proteins; D012191:Retroviridae Proteins; D012996:Solutions; D014452:Ubiquitins; D054311:nef Gene Products, Human Immunodeficiency Virus; C016680:polyacrylamide gels; D014867:Water