title: Double tryptophan exciton probe to gauge proximal side chains in proteins: augmentation at low temperature.
abstract: The circular dichroic (CD) exciton couplet between tryptophans and/or tyrosines offers the potential to probe distances within 10 Å in proteins. The exciton effect has been used with native chromophores in critical positions in a few proteins. Here, site-directed mutagenesis created double tryptophan probes for key sites of a protein (tear lipocalin). For tear lipocalin, the crystal and solution structures are concordant in both apo- and holo-forms. Double tryptophan substitutions were performed at sites that could probe conformation and were likely within 10 Å. Far-UV CD spectra of double Trp mutants were performed with controls that had noninteracting substituted tryptophans. Low temperature (77 K) was tested for augmentation of the exciton signal. Exciton coupling appeared with tryptophan substitutions at positions within loop A-B (28 and 31, 33), between loop A-B (28) and strand G (103 and 105), as well as between the strands B (35) and C (56). The CD exciton couplet signals were amplified 3-5-fold at 77 K. The results were concordant with close distances in crystal and solution structures. The exciton couplets had functional significance and correctly assigned the holo-conformation. The methodology creates an effective probe to identify proximal amino acids in a variety of motifs.
authors.affiliation: Departments of Pathology and Ophthalmology and Jules Stein Eye Institute, University California at Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Oktay K
authors.initials: OK
authors.lastname: Gasymov
authors.forename: Adil R
authors.initials: AR
authors.lastname: Abduragimov
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Glasgow
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D054835:Lipocalin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D054835:Lipocalin 1; D014364:Tryptophan