SITE-DIRECTED DICHROISM AS A METHOD FOR OBTAINING ROTATIONAL AND ORIENTATIONAL CONSTRAINTS FOR ORIENTED POLYMERS

We present the theory of site-directed dichroism and its application t o the determination of rotational and orientational constraints for or iented polypeptides such as transmembrane helices. Infrared spectrosco py dichroism measurements of single amide I vibrational modes correspo nding to C-13-labeled sites within the polypeptide contain information about the helix tilt and rotation angles. This information is readily extracted by analysis of the dichroism of a set of sites along the pe ptide sequence; Data for just two consecutive sites in the dimeric tra nsmembrane domain of glycophorin A yield the tilt of the helix axis wi th respect the membrane normal and the rotation of the helix about its axis. By using dichroism data from three consecutive sites, the helix orientation parameters and the orientation of the amide I transition dipole moment, a, can be obtained; the parameters are in close agreeme nt with the solution NMR structure of the glycophorin A peptide dimer and literature values for a. The approach provides orientational infor mation about selectively labeled peptides even under conditions of mod est fractional sample order.