title: Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin.
abstract: Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.
authors.affiliation: Department of Physics and Astronomy, SUNY at Stony Brook, Stony Brook, NY 11794-5115, USA.
authors.forename: Evan
authors.initials: E
authors.lastname: Crocker
authors.forename: Ashish B
authors.initials: AB
authors.lastname: Patel
authors.forename: Markus
authors.initials: M
authors.lastname: Eilers
authors.forename: Shobini
authors.initials: S
authors.lastname: Jayaraman
authors.forename: Elena
authors.initials: E
authors.lastname: Getmanova
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: Martine
authors.initials: M
authors.lastname: Ziliox
authors.forename: H Gobind
authors.initials: HG
authors.lastname: Khorana
authors.forename: Mordechai
authors.initials: M
authors.lastname: Sheves
authors.forename: Steven O
authors.initials: SO
authors.lastname: Smith
mesh_terms: D000818:Animals; D002244:Carbon / Q000737:chemistry; D002417:Cattle; D002460:Cell Line; D018360:Crystallography, X-Ray; D005998:Glycine / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D011522:Protons; D014443:Tyrosine; D002244:Carbon; D014364:Tryptophan; D012243:Rhodopsin; D005998:Glycine