TY - JOUR
T1 - Characterization of denatured proteins using residual dipolar couplings.
AU - Gebel, Erika B.
AU - Shortle, David
PY - 2007
Y1 - 2007
N2 - The conventional view of denatured proteins as random coils is being challenged by nuclear magnetic resonance (NMR) experiments that can measure the relative orientation of backbone segments with respect to each other in a distance-independent manner. Known as the residual dipolar coupling (RDC), this NMR parameter is measurable on proteins that have been weakly aligned inside a NMR tube. The simple observation of dipolar couplings unequivocally establishes that a denatured protein is not a random coil. Structures of denatured states under differing experimental conditions can be compared by a simple scatter plot of the RDCs measured under each condition; direct structural interpretation, however, is much more problematic. Here some of the technical issues for achieving weak alignment of denatured proteins are addressed using three types of media: strained polyacrylamide gels, both neutral or highly charged, and neutral lipid liquid crystalline bicelles. All three alignment media are experimentally robust and compatible with 8 M urea and low pH, conditions frequently used to denature proteins.
AB - The conventional view of denatured proteins as random coils is being challenged by nuclear magnetic resonance (NMR) experiments that can measure the relative orientation of backbone segments with respect to each other in a distance-independent manner. Known as the residual dipolar coupling (RDC), this NMR parameter is measurable on proteins that have been weakly aligned inside a NMR tube. The simple observation of dipolar couplings unequivocally establishes that a denatured protein is not a random coil. Structures of denatured states under differing experimental conditions can be compared by a simple scatter plot of the RDCs measured under each condition; direct structural interpretation, however, is much more problematic. Here some of the technical issues for achieving weak alignment of denatured proteins are addressed using three types of media: strained polyacrylamide gels, both neutral or highly charged, and neutral lipid liquid crystalline bicelles. All three alignment media are experimentally robust and compatible with 8 M urea and low pH, conditions frequently used to denature proteins.
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M3 - Article
C2 - 16957316
AN - SCOPUS:36849066844
SN - 1064-3745
VL - 350
SP - 39
EP - 48
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -