Polarized FTIR spectroscopy in conjunction with in situ H/D exchange reveals the orientation of protein internal carboxylic acids.
Polarized Fourier transform infrared (FTIR) difference spectroscopy has been combined with in situ H/D exchange measurements to investigate the orientation of single protonated carboxylic acids within bacteriorhodopsin (bR), exclusively based on the protein ground state. The combination of these two techniques enables the determination of the C=O dipole moment direction of D115 and D96 relative to the membrane plane to 68 +/- 11 and 45 +/- 4 degrees , respectively. By discussing these results in the context of X-ray structure analysis, we are able to determine which of the two oxygen atoms of the respective carboxylic acids binds the proton. In the case of D115, it is the oxygen which is located close to T90. On the basis of this finding, we show a possible interaction path between D115 and D85, which has been proposed to be responsible for the inhibition of the proton pump efficiency to prevent over-acidification of the external medium known as the back-pressure effect. Because the orientation of carboxylic acids can be determined even when the group does not undergo any protonation changes during the photocycle, as shown in the case of D115, the method can be applied to any orientable protein and is not merely restricted to bR.
Garczarek
Florian
F
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Gerwert
Klaus
K
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D002264:Carboxylic Acids; D012545:Schiff Bases; D001436:Bacteriorhodopsins