Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin.
Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire.
Sonar
S
S
Physics Department, Boston University, Massachusetts 02215, USA.
Lee
C P
CP
Coleman
M
M
Patel
N
N
Liu
X
X
Marti
T
T
Khorana
H G
HG
RajBhandary
U L
UL
Rothschild
K J
KJ
D001436:Bacteriorhodopsins / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics; D002474:Cell-Free System; D003062:Codon; D003903:Deuterium; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D012365:RNA, Transfer, Tyr / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013462:Sulfur Radioisotopes; D013489:Suppression, Genetic; D014316:Tritium; D014443:Tyrosine / Q000737:chemistry
D003062:Codon; D012365:RNA, Transfer, Tyr; D013462:Sulfur Radioisotopes; D014316:Tritium; D014443:Tyrosine; D001436:Bacteriorhodopsins; D003903:Deuterium