Liganded Hemoglobin Structural Perturbations by the Allosteric Effector L35

Lalezari, I.; Nagel, Ronald L.; Hirsch, Rhoda Elison

doi:10.1529/biophysj.104.046136

Cited by 25 publications

(18 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The extension to proteins via binding to HbA is a significant advance that permits the probing of the hydration shell at a functionally important site of a well studied protein. This advance is possible by virtue of the DPG-like binding properties of HPT (21)(22)(23)(24)(25) and the current finding that the wavelength of the fluorescence emission spectrum of HPT is sensitive to whether the HPT is bound to HbA or free in solution. This latter feature is important in providing assurance that for the HbA containing samples, the reported changes in the polyol/water ratio originate from HbA-bound HPT and not HPT that was released subsequent to the addition of reagents.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study this sensitivity is exploited to probe: (i) the extent to which added glycerol invades the hydration shell of both free and HbA-bound HPT, and (ii) modulation of this effect as a function of added osmolytes including PEG, urea, trehalose and Mg 2ϩ . The potential utility of the HPT fluorescence as a probe of hydration is further enhanced by the observation that HPT, a known fluorescent analog of DPG, binds stoichiometrically to the DPG binding site of human adult hemoglobin (HbA), which is located at the ␤␤ terminus of the water-filled central cavity running through the central core of the ␣ 1 ␤ 1 /␣ 2 ␤ 2 HbA tetramer (21)(22)(23)(24)(25). This site is on the periphery of the protein, where the bound HPT is in contact with the hydration shell waters of HbA.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Molecular Level Probing of Preferential Hydration and Its Modulation by Osmolytes through the Use of Pyranine Complexed to Hemoglobin

Roche

Guo

Friedman

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.It is becoming ever more apparent that water plays a major role both in stabilizing protein structures and in modulating protein dynamics (1-15). Many of the impressive advances in understanding the interplay between water and proteins and the resulting impact on protein stability have been derived from thermodynamic analyses and simulations (14,16,17). Molecular level probing of the hydration shell of proteins under conditions that either enhance or destabilize native structures has proven to be a more daunting task due to the relative dearth of suitable probes and probe techniques. In the present study we utilize pyranine (HPT) 2 as a site-specific fluorescent probe that provides direct spectroscopic detail relating to interactions within hydration layers. In addition, the hydration shell information provided by pyranine is supplemented with Gd 3ϩ vibronic side band data that expose how hydrogen bonding between first and second hydration shell waters is influenced by osmolytes. Together the results from these two probes directly relate to issues such as preferential hydration and osmolyteinduced changes in protein stability. The results are consistent with a relatively simple model that partitions waters and the associated water interactions into three domains: surface waters directly interacting with the hydrated molecule (e.g. protein, cation, chelate etc.), waters within the hydration layer that are impacted by these surface waters, and bulk solvent. The results also support the concept of at least two modes through which osmolytes can alter water interactions within the hydration layer: traditional preferential hydration mechanisms and alteration of hydrogen bonding patterns.The fluorescence from pyranine (8-hydroxy-1,3,6-pyrene trisulfonate, referred to as HPT in the subsequent text) is highly responsive to the composition of its solvent shell. This sensitivity arises from the dissociation/recombination behavior of the single ionizable hydroxyl on the HPT fluorophore (18,19). For the electronic ground state of HPT, the pK a of this prot...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular Level Probing of Preferential Hydration and Its Modulation by Osmolytes through the Use of Pyranine Complexed to Hemoglobin

Roche

Guo

Friedman

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Coletta et al (40), Scott et al (41), and Chen et al (42) have described the liganded Hb with bound organic phosphate as "an altered R-state Hb" or a tertiary T-like conformation within a R-like quaternary structure. A frustrated, unstable R structure (4) has been proposed for HbCO A with IHP bound.…”

Section: Spring-on-scissors Modelmentioning

confidence: 99%

Effector-Induced Structural Fluctuation Regulates the Ligand Affinity of an Allosteric Protein: Binding of Inositol Hexaphosphate Has Distinct Dynamic Consequences for the T and R States of Hemoglobin

Song

Simplăceanu

et al. 2008

Biochemistry

View full text Add to dashboard Cite

The present study reports distinct dynamic consequences for the T-and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 °C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy-and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the ·s-ms time scale, with the exception of several amino acid residues, that is, · 109Val and · 132Lys in deoxy-Hb A, and R40Lys in HbCO A. The mobility of R40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between R40Lys and · 146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of ·109Val, which is located in the intradimer (R 1 · 1 or R 2 · 2 ) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify R40Lys in HbCO A, but does not significantly affect the flexibility of · 109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (R 1 · 2 or R 2 · 1 ) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal ·92His in the ·-heme pocket, as well as some other residues located in the flexible joint (·C helix-RFG corner) and switch (RC helix-·FG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the ·-chain. In contrast, no significant perturbation of the dynamic features on the ms-·s time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the ·-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate (R ex ) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.

show abstract

“…13 Two binding sites were suggested by the published fluorescence data. In the presence of IHP, the drop in fluorescence intensity was more pronounced; IHP caused the high and low affinity sites to raise their L35 affinity by 44% and 10%, respectively.…”

Section: Mechanism Of Reduced Oxygen Affinitymentioning

confidence: 99%

“…12 Recently a detailed spectroscopic study of L35 binding to COHbA has been published, suggesting that L35 induces specific tertiary structural changes within a global R quaternary structure. 13 Several crystal structures of T state Hb bound to L35 and related effectors have been published, but these are at low resolution and not deposited in the Protein Data Bank (PDB). 11 RSR-13 is another artificial effector, very closely related to L35 (two chlorine atoms are replaced by methyl groups, and -NH-by a methylene group).…”

Section: Introductionmentioning

confidence: 99%

R-state Haemoglobin with Low Oxygen Affinity: Crystal Structures of Deoxy Human and Carbonmonoxy Horse Haemoglobin Bound to the Effector Molecule L35

Yokoyama

Neya

Tsuneshige

et al. 2006

Journal of Molecular Biology

View full text Add to dashboard Cite

Liganded Hemoglobin Structural Perturbations by the Allosteric Effector L35

Cited by 25 publications

References 68 publications

Molecular Level Probing of Preferential Hydration and Its Modulation by Osmolytes through the Use of Pyranine Complexed to Hemoglobin

Molecular Level Probing of Preferential Hydration and Its Modulation by Osmolytes through the Use of Pyranine Complexed to Hemoglobin

Effector-Induced Structural Fluctuation Regulates the Ligand Affinity of an Allosteric Protein: Binding of Inositol Hexaphosphate Has Distinct Dynamic Consequences for the T and R States of Hemoglobin

R-state Haemoglobin with Low Oxygen Affinity: Crystal Structures of Deoxy Human and Carbonmonoxy Horse Haemoglobin Bound to the Effector Molecule L35

Contact Info

Product

Resources

About