Modulation of Reactivity and Conformation within the T-Quaternary State of Human Hemoglobin: The Combined Use of Mutagenesis and Sol-Gel Encapsulation
详细信息 查看全文
- 作者：Uri Samuni ; Camille J. Roche ; David Dantsker ; Laura J. Juszczak ; and Joel M. Friedman
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：March 7, 2006
- 年：2006
- 卷：45
- 期：9
- 页码：2820 - 2835
- 全文大小：406K
- 年卷期：v.45,no.9(March 7, 2006)
- ISSN：1520-4995
文摘
A range of conformationally distinct functional states within the T quaternary state ofhemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation thatgreatly slow or eliminate the T 
R transition. Visible and UV resonance Raman spectroscopy are usedto probe the proximal strain at the heme and the status of the 
1
beta2.gif" BORDER=0 ALIGN="middle">2 interface, respectively, whereas COgeminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method)analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutantsused in this study are Hb(Nbeta2.gif" BORDER=0 ALIGN="middle">102A) and the 99-99 cross-linked derivative of Hb(Wbeta2.gif" BORDER=0 ALIGN="middle">37E). The formermutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligatedT-state conformations, whereas the latter mutant is used to access the high-affinity end of the distributionof T-state conformations. A pattern emerges within the T state in which ligand reactivity increases asboth the proximal strain and the 1beta2.gif" BORDER=0 ALIGN="middle">2 interface interactions are progressively lessened after ligand bindingto the deoxy T-state species. The ligation and effector-dependent interplay between the heme environmentand the stability of the Trp beta2.gif" BORDER=0 ALIGN="middle">37 cluster in the hinge region of the 1beta2.gif" BORDER=0 ALIGN="middle">2 interface appears to determine thedistribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented,suggesting that different T quaternary structures modulate the stability of different beta2.gif" BORDER=0 ALIGN="middle"> dimer conformationswithin the tetramer.
R transition. Visible and UV resonance Raman spectroscopy are usedto probe the proximal strain at the heme and the status of the 1beta2.gif" BORDER=0 ALIGN="middle">2 interface, respectively, whereas COgeminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method)analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutantsused in this study are Hb(Nbeta2.gif" BORDER=0 ALIGN="middle">102A) and the 99-99 cross-linked derivative of Hb(Wbeta2.gif" BORDER=0 ALIGN="middle">37E). The formermutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligatedT-state conformations, whereas the latter mutant is used to access the high-affinity end of the distributionof T-state conformations. A pattern emerges within the T state in which ligand reactivity increases asboth the proximal strain and the 1beta2.gif" BORDER=0 ALIGN="middle">2 interface interactions are progressively lessened after ligand bindingto the deoxy T-state species. The ligation and effector-dependent interplay between the heme environmentand the stability of the Trp beta2.gif" BORDER=0 ALIGN="middle">37 cluster in the hinge region of the 1beta2.gif" BORDER=0 ALIGN="middle">2 interface appears to determine thedistribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented,suggesting that different T quaternary structures modulate the stability of different beta2.gif" BORDER=0 ALIGN="middle"> dimer conformationswithin the tetramer.