Isotope-Edited Infrared Linear Dichroism:  Determination of Amide Orientational Relationships

A new approach for the determination of local amide orientation in ordered insoluble proteins using linearly-polarized infrared radiation is described. The method was applied to the crystalline peptide cleromyrine (cyclo(GlyProLeuProGlyTyr)). Labeling of individual amide carbonyl carbons with 13 C resulted in the frequency shift of the affected vibrational mode. 13 C labeling allowed the amide I modes to by systematically assigned (“isotope editing”). Subsequently, the absorption of linearly-polarized infrared radiation was measured in order to determine the orientation of the individual amide carbonyl relative to the incident radiation (infrared linear dichroism). The relative orientations of four cleromyrine amide carbonyls determined in this way were within 15° of those measured from the crystal structure. This method may be useful for the determination of amide orientation in peptides and proteins that form oriented insoluble aggregates that are resistant to crystallization.