Functional and Spectroscopic Characterization of Half-Liganded Iron−Zinc Hybrid Hemoglobin:  Evidence for Conformational Plasticity within the T State<sup>,</sup>

Samuni, Uri; Juszczak, Laura J.; Dantsker, David; Khan, Imran; Friedman, Adam; Pérez-González-de-Apodaca, José; Bruno, Stefano; Hui, Hilda L.; Colby, Judith; Karasik, Ellen; Kwiatkowski, Laura D.; Mozzarelli, Andrea; Noble, Robert W.; Friedman, Joel M.

doi:10.1021/bi020648j

Cited by 47 publications

(96 citation statements)

References 82 publications

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“…A structural basis for the linkage between heme reactivity and both effector and ligation-dependent distributions of HT and LT within the T-state can be seen from previous spectroscopic earlier works (20,21,26,62,63). These UV and visible resonance Raman studies on T-state populations with different HT/LT composition show that the highly stable T-state ␣ 1 ␤ 2 interface associated with deoxy-HbA undergoes progressive loosening in going from the extreme LT to HT populations.…”

Section: Discussionmentioning

confidence: 85%

“…This point is illustrated by the comparison of FeCO/Cr 3ϩ and FeCO/Zn 2ϩ data showing that within the T-state, ligand binding (as mimicked by the Cr 3ϩ ion) biases the HT/LT distribution toward HT with respect to the remaining deoxy sites. The Raman results show that in shifting toward the HT population, proximal strain decreases, which is associated with enhanced ligand reactivity (20,21,27,62). In addition we anticipate that it is also likely that access to the ␤ heme is enhanced due to either a shifting of the Val E11 side chain or to an interface-associated loosening that enhances the amplitude of side chain fluctuations that transiently increase access to the ␤ heme.…”

Section: Discussionmentioning

confidence: 88%

“…The Fe 2ϩ /Zn 2ϩ hybrids are known both to bind oxygen non-cooperatively with very low affinity (31) and to remain in the T-state when liganded under a variety of solution conditions (20). The Zn 2ϩ substitution mimics the behavior of the five coordinate ferrous irons in deoxy-HbA.…”

Section: Sample Characterizationmentioning

confidence: 99%

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Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Roche

Dantsker

Samuni

et al. 2006

Journal of Biological Chemistry

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Nitrite reductase activity of deoxyhemoglobin (HbA) in the red blood cell has been proposed as a non-nitric-oxide synthase source of deliverable nitric oxide (NO) within the vasculature. An essential element in this scheme is the dependence of this reaction on the quaternary/tertiary structure of HbA. In the present work sol-gel encapsulation is used to trap and stabilize deoxy-HbA in either the T or R quaternary state, thus allowing for the clear-cut monitoring of nitrite reductase activity as a function of quaternary state with and without effectors. The results indicate that reaction is not only R-T-dependent but also heterotropic effector-dependent within a given quaternary state. The use of the maximum entropy method to analyze carbon monoxide (CO) recombination kinetics from fully and partially liganded sol-gel-encapsulated T-state species provides a framework for understanding effector modulation of T-state reactivity by influencing the distribution of high and low reactivity T-state conformations.The physiological role of the nitrite ion is currently attracting considerable research interest arising primarily from observations that indicate the anion can function as a non-nitric-oxide synthase source of nitric oxide (NO) (1-6). It has also been suggested that hemoglobin (Hb) 2 within red blood cells may be a source of deliverable nitrite-derived NO, generated from Hb nitrite reductase activity (deoxy-Hb ϩ nitrite 3 met-Hb ϩ NO) (6 -10). Such a mechanism also has clear implications for the vasoactivity of acellular hemoglobin-based blood substitutes. Although there are studies that support the physiological role of a hemoglobin-based source of nitrite-derived NO, there are still fundamental mechanistic questions that remain unanswered including the key issue of how NO, once bound to a ferrous heme, can be efficiently delivered (11, 12).Studies show that there is a relationship between the hemoglobin P50 and nitrite reductase activity (2, 3, 13, 14) as well as S-nitrosothiol synthase function (14). This result has led to the hypotheses that red blood cell/Hb enzymatic pathways can modulate blood flow and play a role in regulating hypoxic vasodilation (3, 14). The results also imply a relationship between the conformational properties of Hb and nitrite reductase activity (2, 5, 6, 15) and S-nitrosothiol synthase function (14). Direct measurements of the nitrite reductase activity of deoxy-Hb as a function of added allosteric effectors and mutagenic/chemical modifications support the concept of conformational control of Hb nitrite reductase reactivity with the T-state conformation having reduced reactivity compared with R-state conformations of Hb (5, 6). Two limitations of these studies are the inability to stabilize and compare the T and R-state forms of deoxy-HbA (human adult hemoglobin) without mutagenic or chemical modification and the complexity of the reductase reaction in solution due to autoacceleration arising from the allosteric nature of Hb reactivity.The present study directly addresses the qu...

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 88%

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Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Roche

Dantsker

Samuni

et al. 2006

Journal of Biological Chemistry

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“…[5][6][7][8][9][10][11][12][13] In addition, tertiary and quaternary conformational changes in heme proteins, such as myoglobin (Mb) and hemoglobin (Hb), can be inhibited or dramatically slowed, which allows these proteins to be "trapped" and studied in non-equilibrium structural conformations. [14][15][16][17][18][19][20][21][22] The origin of the stability imparted by the silica sol-gel matrix is complex. Since the mean pore diameters in aged wet sol-gels (typically <10 nm) are comparable to the diameters of Mb and Hb, the pore walls could physically restrict protein motions.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

2006

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Spectrally-resolved infrared stimulated vibrational echo spectroscopy is used to measure the fast dynamics of heme-bound CO in carbonmonoxy-myoglobin (MbCO) and hemoglobin (HbCO) embedded in silica sol-gel glasses. On the time scale of ~100 fs to several ps, the vibrational dephasing of the heme-bound CO is measurably slower for both MbCO and HbCO relative to aqueous protein solutions. The fast structural dynamics of MbCO, as sensed by the heme-bound CO, are influenced more by the sol-gel environment than those of HbCO. Longer time scale structural dynamics (tens of ps), as measured by the extent of spectral diffusion, are the same for both proteins encapsulated in sol-gel glasses compared to aqueous solutions. A comparison of the sol-gel experimental results to viscosity dependent vibrational echo data taken on various mixtures of water and fructose shows that the sol-gel encapsulated MbCO exhibits dynamics that are the equivalent to the protein in a solution that is nearly 20 times more viscous than bulk water. In contrast, the HbCO dephasing in the sol-gel reflects only a 2-fold increase in viscosity. Attempts to alter the encapsulating pore size by varying the molar ratio of silane precursor to water (R-value) used to prepare the sol-gel glasses were found to have no effect on the fast or steady-state spectroscopic results. The vibrational echo data are discussed in the context of solvent confinement and protein-pore wall interactions to provide insights into the influence of a confined environment on the fast structural dynamics experienced by a biomolecule.

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“…In particular, it has been shown that after photolysis of HbCO encapsulated in the T state, both the E and F helices assume intermediate positions with respect to those that characterize the Hb equilibrium states. Moreover, ligand rebinding kinetics of CO to encapsulated Hb were measured in flash photolysis experiments both at room [58,87,89,90,127,[129][130][131][132] and cryogenic temperatures [88]. Inhibition of the quaternary transition allowed to study CO rebinding kinetics to pure T and R states.…”

Section: Trapping Reaction Intermediates: Inhibition Of Conformationamentioning

confidence: 99%

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

Ronda

Bruno

Campanini

et al. 2015

COC

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Abstract:The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques.

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Functional and Spectroscopic Characterization of Half-Liganded Iron−Zinc Hybrid Hemoglobin: Evidence for Conformational Plasticity within the T State^,

Cited by 47 publications

References 82 publications

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

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Functional and Spectroscopic Characterization of Half-Liganded Iron−Zinc Hybrid Hemoglobin: Evidence for Conformational Plasticity within the T State,

Cited by 47 publications

References 82 publications

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

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Product

Resources

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Functional and Spectroscopic Characterization of Half-Liganded Iron−Zinc Hybrid Hemoglobin: Evidence for Conformational Plasticity within the T State^,