Tyrosine and Tryptophan Structure Markers in Hemoglobin Ultraviolet Resonance Raman Spectra: Mode Assignments via Subunit-Specific Isotope Labeling of Recombinant Protein
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- 作者：Xuehua Hu and Thomas G. Spiro
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：December 16, 1997
- 年：1997
- 卷：36
- 期：50
- 页码：15701 - 15712
- 全文大小：396K
- 年卷期：v.36,no.50(December 16, 1997)
- ISSN：1520-4995
文摘
Phenyl-deuterated tyrosine (Tyr-d4) andindole-deuterated tryptophan (Trp-d5) havebeenselectively incorporated into hemoglobin (Hb) by expressing the gene inauxotrophic strains of Escherichiacoli. Ultraviolet resonance Raman (UVRR) spectra, using229-nm excitation, show that difference featurescharacteristic of the Hb quaternary R 
mages/entities/rarr.gif"> T transition are notperturbed by the incorporation of the isotopes.All the UVRR bands between 800 and 1700 cm-1 areassigned to either Tyr or Trp except for the 1511cm-1 band, which had been thought to arise from the Trp 2× W18 overtone. This band does not shiftupon Trp or Tyr labeling but does shift 5 cm-1 inD2O, suggesting assignment to a histidine (His)residue.Its intensification in the T-state is consistent with Hisprotonation. The mages/gifchars/alpha.gif" BORDER=0>- and mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-subunits wereselectivelylabeled, by reconstitution of labeled subunits with unlabeled subunits,to make isotope hybrids. SelectiveTyr labeling identified the mages/gifchars/alpha.gif" BORDER=0> subunits as the locus of the Y8aupshift observed in Hb, supporting theprevious inference that this shift is associated with the T-stateH-bond involving the interfacial Tyr mages/gifchars/alpha.gif" BORDER=0>42[Rodgers, Su, Subramaniam, & Spiro (1992) J. Am.Chem. Soc. 114, 3697]. SelectiveTrp labeling showedthe Trp mages/gifchars/alpha.gif" BORDER=0>14 contributions to the T - R difference spectrum to benegligible and confirmed Trp mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">37 asthe locus of the W3 difference signal, and probably of the remainingTrp signals as well. The observeddownshift of W17 and upshift of Wd5 in the T-state are consistent witha stronger T-state H-bond betweenTrp mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">37 and Asp mages/gifchars/alpha.gif" BORDER=0>94; the resulting excitation profile red shiftaccounts for the dominance of the Trp mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">37contribution to the T - R difference UVRR spectrum.