Hydrogen-deuterium exchange in membrane proteins monitored by IR spectroscopy: a new tool to resolve protein structure and dynamics.
As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record (1)H/(2)H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H(+),K(+)-ATPase, demonstrates the usefulness of (1)H/(2)H exchange kinetics for the understanding of the molecular movement related to the catalytic activity.
Vigano
C
C
Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Free University of Brussels, CP 206/2, Boulevard du Triomphe, B-1050 Brussels, Belgium.
Smeyers
M
M
Raussens
V
V
Scheirlinckx
F
F
Ruysschaert
J M
JM
Goormaghtigh
E
E
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D002151:Calorimetry; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D003903:Deuterium; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry; D006859:Hydrogen; D007700:Kinetics; D008956:Models, Chemical; D015233:Models, Statistical; D016011:Normal Distribution; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D013055:Spectrophotometry, Infrared / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared; D013270:Stomach / Q000201:enzymology; D013997:Time Factors; D014867:Water
D011522:Protons; D014867:Water; D006859:Hydrogen; D000255:Adenosine Triphosphate; D003903:Deuterium; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase