Conformational Changes in FmetHbS Probed with UV Resonance Raman and Fluorescence Spectroscopic Methods

Sokolov, L.I.; Mukerji, Ishita

doi:10.1021/jp982099q

Cited by 13 publications

(26 citation statements)

References 31 publications

(79 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The change in intensity at the 1558 cm Ϫ1 band assigned to differences in the H-bonding between ␤15 Trp and ␤72 Ser of the Aand E-helices may be predictive of alterations in the DPG pocket. Similar UVRR findings for the T-state fluromet HbS were reported recently (48).…”

Section: Discussionsupporting

confidence: 90%

“…Our results point to A-helix alterations that may be the likely candidate in the primary mechanism driving oxy HbC to crystallize and causing destabilization of oxy HbS, which, under deoxy conditions, may give rise to the polymerization process. Similar A-helix perturbations have also been reported by others for both T-and R-state forms of fluoro-met HbS (48). Extensive comparative investigations of the deoxy forms of these mutants are planned.…”

Section: Discussionsupporting

confidence: 79%

See 1 more Smart Citation

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

Hirsch¹,

Juszczak²,

Fataliev³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the ␣ and ␤ chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC.We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the ␣ 1 ␤ 2 interface (␤37), and in the A-helix (␣14/␤15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC ϳ HbS > HbA.Naturally occurring ␤6 hemoglobin mutants aggregate into defined structures in the erythrocyte. Sickle cell hemoglobin (HbS, ␤6 Glu 3 Val) forms polymers in the deoxy state, whereas HbC (␤6 Glu 3 Lys) forms crystals in the oxy liganded state. A complete understanding of the mechanisms giving rise to deoxy HbS polymers and oxy HbC crystals remains to be elucidated. In the last few years, our laboratory has pursued questions related to mechanisms involved in the ligand-specific, induced crystallization of HbC, starting with site-specific probing of the R-state tetrameric structure of HbC (1, 2). Here, we extend the studies to include a comparison of the R-state of HbS. The R-state of HbS is of particular relevance because oxy HbS exhibits unusual properties compared with HbA, such as mechanical precipitability (3-5), greater unfolding at an airwater interface (6, 7), and increased autooxidation (8 -10).According to existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, this ignores the following: 1) important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the ␣ and ␤ chains), and 2) crystals might constrain conformation compared with that in solution.Spectroscopic and biochemical findin...

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 79%

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

Hirsch¹,

Juszczak²,

Fataliev³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…It is not clear whether changes in hydration induce slight changes in the ␣ helix itself. The ␣14 and ␤15 tryptophan residues are not distinguishable by UVRR spectroscopy, but given that the mutations studied here are located on the ␤ chain, all of the differences observed on the W3 ␣14␤15 main band can be reasonably assigned to ␤Trp 15 (14). ␤Trp 15 is an A helix residue situated in the crevice formed by the A and E helix.…”

Section: R 3 T State Transition-associated Spectral Changesmentioning

confidence: 78%

Conformational Changes in Hemoglobin S (βE6V) Imposed by Mutation of the βGlu7–βLys132 Salt Bridge and Detected by UV Resonance Raman Spectroscopy

Juszczak¹,

Fablet²,

Baudin‐Creuza³

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, ␤Glu 7 -␤Lys 132 , are dependent on which residue of the pair is modified. The ␤K132A mutants exhibit the spectroscopic signatures of the R 3 T state transition in both the "hinge" and "switch" regions of the ␣ 1 ␤ 2 interface. Both singly and doubly mutated hemoglobin (Hb) ␤⌭7〈 exhibit the switch region signature for the R 3 T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb ␤⌭7〈 mutants. The observed large decrease in the W3 ␣14␤15 band intensity for doubly mutated Hb ␤⌭7〈 is attributed to an enhanced separation in the A helix-E helix tertiary contact of the ␤ subunits. The results for the Hb A ␤Glu 7 -␤Lys 132 salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the ␣ 1 ␤ 2 dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.Sickle cell hemoglobin (Hb S, 1 ␤6(A3) Glu 3 Val) exhibits the property of anomalous and pathologic self-assembly. DeoxyHb S forms polymers in the erythrocyte, which leads to microvascular blockage, organ damage, and often premature death. Structure-based drug design requires knowledge of optimal polymer disruption sites. The specific interaction in the Hb S polymer involves the steric fit of the mutated hydrophobic ␤6 donor site in a hydrophobic acceptor site located in an adjacent Hb S tetramer. It is well known that the hydrophobicity and the stereospecificity of the donor site are essential to the initiation of the polymerization.Lesecq et al.(1, 2) have been investigating whether modification of the polarity close to the ␤6 site could influence the packing of the donor and acceptor sites, thus modifying the polymerization process. Replacing the hydrophilic Glu ␤7(A4) residue with a hydrophobic Ala residue resulted in a decreased polymerization of the doubly mutated rHb ␤E6V/E7A. It was postulated (1, 2) that the loss of the normal salt bridge between ␤Glu 7 (A4) and ␤Lys 132 (H10) in the rHb ␤E7A mutants might lead to an alteration in both the position and the mobility of the A helix, illustrated in Fig. 1. These alterations of the A helix might result in a misfit between the donor and acceptor sites, which could explain the observed diminution in polymerization. It follows from this hypothesis that modifying the other partner of the salt bridge, ␤Lys 132 (H10), should have similar consequences on polymerization (2).Visible resonance Raman spectroscopy is very useful in providing detailed information relating to the influence of tertiary and quaternary structure upon specific heme-related vibrational degr...

show abstract

“…39,40,56,57 The nanosecond pulse duration allows nanosecond time resolution in time-resolved resonance Raman (TR 3 ) or time-resolved temperature-jump UVRR measurements. 39,40,56,57 The nanosecond pulse duration allows nanosecond time resolution in time-resolved resonance Raman (TR 3 ) or time-resolved temperature-jump UVRR measurements.…”

Section: Third Generation Uv-vis Lasers For Uv-vis Rr Studiesmentioning

confidence: 99%

Resonance Raman and surface- and tip-enhanced Raman spectroscopy methods to study solid catalysts and heterogeneous catalytic reactions

et al. 2010

View full text Add to dashboard Cite

Resonance Raman (RR) spectroscopy has several advantages over the normal Raman spectroscopy (RS) widely used for in situ characterization of solid catalysts and catalytic reactions. Compared with RS, RR can provide much higher sensitivity and selectivity in detecting catalytically-significant surface metal oxides. RR can potentially give useful information on the nature of excited states relevant to photocatalysis and on the anharmonic potential of the ground state. In this critical review a detailed discussion is presented on several types of RR experimental systems, three distinct sources of so-called Raman (fluorescence) background, detection limits for RR compared to other techniques (EXAFS, PM-IRAS, SFG), and three well-known methods to assign UV-vis absorption bands and a band-specific unified method that is derived mainly from RR results. In addition, the virtues and challenges of surface-enhanced Raman spectroscopy (SERS) are discussed for detecting molecular adsorbates at catalytically relevant interfaces. Tip-enhanced Raman spectroscopy (TERS), which is a combination of SERS and near-field scanning probe microscopy and has the capability of probing molecular adsorbates at specific catalytic sites with an enormous surface sensitivity and nanometre spatial resolution, is also reviewed (300 references).

show abstract

Conformational Changes in FmetHbS Probed with UV Resonance Raman and Fluorescence Spectroscopic Methods

Cited by 13 publications

References 31 publications

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

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

Conformational Changes in Hemoglobin S (βE6V) Imposed by Mutation of the βGlu7–βLys132 Salt Bridge and Detected by UV Resonance Raman Spectroscopy

Resonance Raman and surface- and tip-enhanced Raman spectroscopy methods to study solid catalysts and heterogeneous catalytic reactions

Contact Info

Product

Resources

About