Mixing apparatus for preparing NMR samples under pressure.
The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.
Wu
Wen-Jin
WJ
Department of Biochemistry, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544, USA.
Vidugiris
Gediminas
G
Mooberry
Ed S
ES
Westler
William M
WM
Markley
John L
JL
D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000295:instrumentation* / Q000379:methods*; D008823:Micelles; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D011312:Pressure; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry*; D014422:Turkeys / Q000378:metabolism; D014783:Viscosity
D008823:Micelles; D011506:Proteins; D012996:Solutions; D002918:Chymotrypsin