Premium
Site‐specific examination of secondary structure and orientation determination in membrane proteins: The peptidic 13 C 18 O group as a novel infrared probe
Author(s) -
Torres Jaume,
Kukol Andreas,
Goodman Jonathan M.,
Arkin Isaiah T.
Publication year - 2001
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/1097-0282(200111)59:6<396::aid-bip1044>3.0.co;2-y
Subject(s) - chemistry , protein secondary structure , isotope , isotopic labeling , macromolecule , infrared , fourier transform infrared spectroscopy , infrared spectroscopy , resolution (logic) , spectroscopy , yield (engineering) , amide , crystallography , analytical chemistry (journal) , organic chemistry , biochemistry , physics , quantum mechanics , artificial intelligence , computer science , optics , materials science , metallurgy
Detailed site‐specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1‐ 13 CO labels have been shown to relay site‐specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of 13 C and the lack of baseline resolution between the main amide I band and the isotope‐edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1‐ 13 C 18 O label. The double‐isotope label virtually eliminates any contribution from natural abundance 13 C. More importantly, the isotope‐edited peak is further red‐shifted (in accordance with ab initio Hartree–Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1‐ 13 C 18 O is far preferable as a label in comparison to 1‐ 13 C 16 O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site‐directed dichroism to the structural elucidation of polytopic membrane proteins. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 396–401, 2001