Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: the "molecular-sandwich" technique.
The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of >/=0.2 nm (approximately 5% of the molecule's height) with a time resolution of >/=1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM "molecular-sandwich" technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.
Oberleithner
H
H
Department of Physiology, University of Münster, Robert-Koch-Strasse 27a Innenhof, D-48149 Münster, Germany. oberlei@uni-muenster.de
Schneider
S W
SW
Henderson
R M
RM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004573:Electrolytes; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods*; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013269:Stochastic Processes
D004573:Electrolytes; C510692:Kcnj1 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D012996:Solutions; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases