title: Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation.
abstract: A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate 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 mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution 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 alphabeta dimer conformations within the tetramer.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Uri
authors.initials: U
authors.lastname: Samuni
authors.forename: Camille J
authors.initials: CJ
authors.lastname: Roche
authors.forename: David
authors.initials: D
authors.lastname: Dantsker
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Juszczak
authors.forename: Joel M
authors.initials: JM
authors.lastname: Friedman
mesh_terms: D002248:Carbon Monoxide / Q000737:chemistry; D005782:Gels / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008024:Ligands; D016296:Mutagenesis; D044367:Phase Transition; D020836:Protein Structure, Quaternary; D012996:Solutions / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship
chemical_list: D005782:Gels; D006454:Hemoglobins; D008024:Ligands; D012996:Solutions; D002248:Carbon Monoxide; C100003:deoxyhemoglobin