Tyrosine and Tryptophan Structure Markers in Hemoglobin Ultraviolet Resonance Raman Spectra:  Mode Assignments via Subunit-Specific Isotope Labeling of Recombinant Protein

Abstract

Phenyl-deuterated tyrosine (Tyr-d4) and indole-deuterated tryptophan (Trp-d5) have been selectively incorporated into hemoglobin (Hb) by expressing the gene in auxotrophic strains of Escherichia coli. Ultraviolet resonance Raman (UVRR) spectra, using 229-nm excitation, show that difference features characteristic of the Hb quaternary R --> T transition are not perturbed by the incorporation of the isotopes. All the UVRR bands between 800 and 1700 cm-1 are assigned to either Tyr or Trp except for the 1511 cm-1 band, which had been thought to arise from the Trp 2 x W18 overtone. This band does not shift upon Trp or Tyr labeling but does shift 5 cm-1 in D2O, suggesting assignment to a histidine (His) residue. Its intensification in the T-state is consistent with His protonation. The alpha- and beta-subunits were selectively labeled, by reconstitution of labeled subunits with unlabeled subunits, to make isotope hybrids. Selective Tyr labeling identified the alpha subunits as the locus of the Y8a upshift observed in Hb, supporting the previous inference that this shift is associated with the T-state H-bond involving the interfacial Tyr alpha42 [Rodgers, Su, Subramaniam, & Spiro (1992) J. Am. Chem. Soc. 114, 3697]. Selective Trp labeling showed the Trp alpha14 contributions to the T - R difference spectrum to be negligible and confirmed Trp beta37 as the locus of the W3 difference signal, and probably of the remaining Trp signals as well. The observed downshift of W17 and upshift of Wd5 in the T-state are consistent with a stronger T-state H-bond between Trp beta37 and Asp alpha94; the resulting excitation profile red shift accounts for the dominance of the Trp beta37 contribution to the T - R difference UVRR spectrum.

Solution-active Structural Alterations in Liganded Hemoglobins C (β6 Glu → Lys) and S (β6 Glu → Val)

“…UVRR difference spectra of hemoglobins typically exhibit two categories of change (37,40,41,42). The first can readily be attributed to quaternary structure-based differences in the ␣ 1 ␤ 2 interface.…”

“…Ultraviolet Resonance Raman Spectroscopy-UVRR spectroscopy of hemoglobins site selectively probes the aromatic residues of the globin domains (35)(36)(37) (35)(36)(37). When the spectra are normalized at the nitrate internal standard as shown (Fig.…”

Raman Spectroscopy of Proteins

“…1 A band of HbA at 1511 cm 1 , which is suggested to be related to Trp, is shifted to a higher frequency by 5 cm 1 in D 2 O. 39 This band has been noted as a quantitative T-specific indicator of HbA. 45 …”

Raman structural markers of tryptophan and histidine side chains in proteins

“…6 An analog of Trp with deuterated ring carbons, Trp-2,4,5,6,7-d 5 (Trp-d 5 ), is sometimes used to label Trp residues in peptides and proteins. [23][24][25][26][27] The deuteration has little affect on the vibrational mode of W3 and the wavenumber of the corresponding vibration of Trp-d 5 (Wd3) also changes with | 2,1 | in a way similar to that of W3.…”

“…5,23 In UV resonance Raman spectra excited at 229 nm, the W4 band can be detected but the W6 band is not clear. 27,38,39 The wavenumbers of the Wd4 (Ϸ1445 cm Ϫ1 ) and Wd5 (Ϸ1405 cm Ϫ1 ) bands of Trp-d 5 also depend on hydrogen bonding in a way similar to that of the W4 and W6 bands of Trp. 8 The Wd4 and Wd5 bands are detectable in 229-nm excited Raman spectra 27 and may be useful in studying the Adapted from the data of Miura et al 5 The atomic displacements in the two vibrations are also shown.…”