title: Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery.
abstract: The near-UV magnetic circular dichroism spectroscopy of the aromatic amino acid bands of hemoglobin was investigated as a potential probe of structural changes at the alpha(1)beta(2) interface during the allosteric transition. Allosteric effectors were used to direct carp and chemically modified human hemoglobins into the R (relaxed) or T (tense) state in order to determine the heme-ligation-independent spectral characteristics of the quaternary states. The tryptophan magnetic circular dichroism (MCD) peak observed at 293 nm in the R state of N-ethylsuccinimide- (NES-) des-Arg-modified human hemoglobin (Hb) was shifted to a slightly longer wavelength in the T state, consistent with the shift expected for tryptophan acting as a proton donor in a T-state hydrogen bond. Moreover, the increase observed in the T-state MCD intensity of this band relative to the R-state intensity was consistent with the effect expected for proton donation by tryptophan on the basis of the Michl perimeter model of aromatic MCD. The peak-to-trough magnitude of the R - T MCD difference spectrum is equal to 30% of the total R-state peak intensity contributed by all six tryptophans present in the human tetramer; the relative magnitude specific to the two beta37 tryptophans undergoing conformational change is estimated accordingly to be 3 times larger. The Trp-beta37 spectral shift, about 200 cm(-)(1), is in good agreement with the shifts observed in other H-bonded proton donors and provides corroborating spectral evidence for the formation in solution of a T-state Trp beta37-Asp alpha94 hydrogen bond observed in X-ray diffraction studies of deoxyHb crystals.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Vitale
authors.forename: R A
authors.initials: RA
authors.lastname: Goldbeck
authors.forename: D B
authors.initials: DB
authors.lastname: Kim-Shapiro
authors.forename: R M
authors.initials: RM
authors.lastname: Esquerra
authors.forename: L J
authors.initials: LJ
authors.lastname: Parkhurst
authors.forename: D S
authors.initials: DS
authors.lastname: Kliger
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002347:Carps; D002942:Circular Dichroism; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011487:Protein Conformation; D013388:Succinimides; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D013388:Succinimides; D014364:Tryptophan