Near-ultraviolet magnetic circular dichroism spectroscopy of protein conformational states: correlation of tryptophan band position and intensity with hemoglobin allostery.
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.
Vitale
D J
DJ
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Goldbeck
R A
RA
Kim-Shapiro
D B
DB
Esquerra
R M
RM
Parkhurst
L J
LJ
Kliger
D S
DS
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*
D006454:Hemoglobins; D013388:Succinimides; D014364:Tryptophan