Site-specific examination of secondary structure and orientation determination in membrane proteins: the peptidic (13)C=(18)O group as a novel infrared probe.
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)C=(18)O 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=(18)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.
Torres
J
J
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Kukol
A
A
Goodman
J M
JM
Arkin
I T
IT
D001704:Biopolymers / Q000737:chemistry; D002247:Carbon Isotopes; D008565:Membrane Proteins / Q000737:chemistry*; D010103:Oxygen Isotopes; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
D001704:Biopolymers; D002247:Carbon Isotopes; D008565:Membrane Proteins; D010103:Oxygen Isotopes