Tyrosine and tryptophan structure markers in hemoglobin ultraviolet resonance Raman spectra: Mode assignments via subunit-specific isotope labeling of recombinant protein
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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DOI：
10.1021/bi971136l
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发表时间：
1997-12-16
期刊：
影响因子：
2.9
通讯作者：
Spiro, TG
中科院分区：
文献类型：
--
作者：
Hu, XH;Spiro, TG
通过在大肠杆菌的营养缺陷型菌株中表达苯基-氘代酪氨酸（Tyr-d（4））和吲哚-氘代色氨酸（Trp-d（5）），已经选择性地将该基因掺入血红蛋白（Hb）中。紫外共振拉曼（UVRR）光谱，使用229 nm的激发，表明Hb四元R->T跃迁的差异特征不受干扰的同位素的掺入。所有在800和1700 cm（-1）之间的UVRR波段都被指定为Tyr或Trp，除了1511 cm（-1）波段，该波段被认为是由Trp 2 x W18泛音产生的。该条带在Trp或Tyr标记后不发生位移，但在D2 O中发生位移5 cm（-1），表明归属于组氨酸（His）残基。它在T状态下的强化与他的质子化是一致的。通过将标记的亚基与未标记的亚基重组，选择性地标记α-和β-亚基，以制备同位素杂合体。选择性Tyr标记将Ct亚基鉴定为在Hb中观察到的Y8 a上移的基因座，支持先前的推断，即该移位与涉及界面Tyr α 42的T-状态H-键相关[Rodgers，Su，Subramaniam，& Spiro（1992）J. Am.化学分析114、3697]。选择性Trp标记显示Trp α 14对T-R差异光谱的贡献可忽略不计，并确认Trp β 37为W3差异信号的位点，并且可能也是剩余Trp信号的位点。观察到的W17的下移和Wd 5在T状态的上移与Trp β 37和Asp α 94之间较强的T状态H键一致;由此产生的激发曲线红移解释了Trp β 37对T-R差异UVRR光谱的主导作用。
Phenyl-deuterated tyrosine (Tyr-d(4)) and indole-deuterated tryptophan (Trp-d(5)) 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 ct 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 alpha 42 [Rodgers, Su, Subramaniam, & Spiro (1992) J. Am. Chem. Sec. 114, 3697]. Selective Trp labeling showed the Trp alpha 14 contributions to the T-R difference spectrum to be negligible and confirmed Trp beta 37 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 beta 37 and Asp alpha 94; the resulting excitation profile red shift accounts for the dominance of the Trp beta 37 contribution to the T-R difference UVRR spectrum.