Monique Laberge

Protein dynamics explain the allosteric behaviors of hemoglobin.

Bohr, Hasselbalch, and Krogh discovered homotropic and heterotropic allosteric behaviors of hemoglobin (Hb) in 1903/1904. A chronological description since then of selected principal models of the allosteric mechanism of Hb, such as the Adair scheme, the MWC two-state concerted model, the KNF induced-fit sequential model, the Perutz stereochemical model, the tertiary two-state model, and the global allostery model (an expanded MWC models), is concisely presented, followed by analysis and discussion of their limitations and deficiencies. The determination of X-ray crystallographic structures of deoxy- and ligated-Hb and the structure-based stereochemical model by Perutz are an epoch-making event in this history. However, his assignment of low-affinity deoxy- and high-affinity oxy-quaternary structures of Hb to the T- and R-states, respectively, though apparently reasonable, and as well as his hypothesis that the T-/R-quaternary structural transition regulates the oxygen-affinity, have created confusions and side-tracked studies of Hb on the structure-function relationship. The differences in static molecular structures of Hb between T(deoxy)- and R(oxy)-quaternary states reported in detail by Perutz and others are ligation-linked structural changes, but not related to the control/regulation of the oxygen-affinity. The oxygen-affinity (K(T) and K(R)) of Hb has been shown to be regulated by the heterotropic effector-linked tertiary structural changes without involving the T/R-quaternary changes. However, a recent high-resolution crystallographic analysis of Hb with different oxygen-affinities shows that static molecular structures of Hb determined by crystallography can neither identify the nature of the T(low-affinity) functional state nor decipher the mechanism by which Hb stores free energy in the T(low-affinity) functional state. Molecular dynamics simulations show that fluctuations of helices of oxy-Hb are increased upon de-oxygenation and/or binding 2,3-biphosphoglycerate. These are known to lower the oxygen-affinity of Hb. It is proposed that the coordination mode of the heme Fe with proximal and distal His is modulated by these helical fluctuations, resulting in the modulation of the oxygen-affinity of Hb. Therefore, it is proposed that the oxygen-affinity of Hb is regulated by pentanary (the 5th-order time-dependent or dynamic) tertiary structural changes rather than the T-/R-quaternary structural transitions in Hb. Homotropic and heterotropic allosteric effects of Hb are oxygen- and effector-linked, conformational entropy-driven entropy-enthalpy compensation phenomena and not much to do with static structural changes. The dynamic allostery model, which integrates these observations, provides the structural basis for the global allostery model (an expanded MWC model).

Intrinsic protein electric fields: basic non-covalent interactions and relationship to protein-induced Stark effects.

Knowledge of the interactions involving charged, polar and polarizable groups in proteins is fundamental, not only because they are important determinants for gaining insight into biophysical molecular recognition and assembly processes, but also for understanding how the matrix of a protein can be viewed as an electric field capable of inducing Stark perturbations on the spectral properties of biological optical centers. This review describes the essential features of noncovalent interactions in protein systems and discusses the concept of the dielectric constant of a protein in the context of different microscopic and macroscopic modeling approaches. It also provides an account of a specific type of high resolution vibrational and optical Stark spectroscopy attempting to correlate the observed spectral properties of biological optical centers to the intrinsic protein fields induced by the matrix in which they reside.

The Endogenous Calcium Ions of Horseradish Peroxidase C Are Required to Maintain the Functional Nonplanarity of the Heme

Horseradish peroxidase C (HRPC) binds 2 mol calcium per mol of enzyme with binding sites located distal and proximal to the heme group. The effect of calcium depletion on the conformation of the heme was investigated by combining polarized resonance Raman dispersion spectroscopy with normal coordinate structural decomposition analysis of the hemes extracted from models of Ca(2+)-bound and Ca(2+)-depleted HRPC generated and equilibrated using molecular dynamics simulations. Results show that calcium removal causes reorientation of heme pocket residues. We propose that these rearrangements significantly affect both the in-plane and out-of-plane deformations of the heme. Analysis of the experimental depolarization ratios are clearly consistent with increased B(1g)- and B(2g)-type distortions in the Ca(2+)-depleted species while the normal coordinate structural decomposition results are indicative of increased planarity for the heme of Ca(2+)-depleted HRPC and of significant changes in the relative contributions of three of the six lowest frequency deformations. Most noteworthy is the decrease of the strong saddling deformation that is typical of all peroxidases, and an increase in ruffling. Our results confirm previous work proposing that calcium is required to maintain the structural integrity of the heme in that we show that the preferred geometry for catalysis is lost upon calcium depletion.

R-state hemoglobin bound to heterotropic effectors: models of the DPG, IHP and RSR13 binding sites

We performed a docking study followed by a 500‐ps molecular dynamics simulation of R‐state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3‐diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2‐[4‐[(3,5‐dichlorophenylcarbamoyl)‐]methyl]‐phenoxy]‐2‐methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T‐state HbA, the effector binding sites are also located in the central cavity of the R‐state and differ depending on effector anionic character. DPG and IHP bind between the α‐subunits and the RSR13 site spans the α1‐, α2‐ and β2‐subunits. The generated models provide the first report of the molecular details of R‐state HbA bound to heterotropic effectors.

Fluorescence line narrowing applied to the study of proteins

Fluorescence line narrowing is a high resolution spectroscopic technique that uses low temperature and laser excitation to optically select specific subpopulations from the inhomogeneously broadened absorption band of the sample. When applied to the study of fluorescent groups in proteins one can obtain vibronically resolved spectra, which can be analyzed to give information on spectral line shapes, vibrational energies of both the ground and excited state molecule, and the inhomogeneous distribution function of the electronic transitions. These parameters reveal information about the chromophoric prosthetic group and the protein matrix and are functions of geometric strains and local electric fields imposed by the protein. Examples of the use of fluorescence line narrowing are discussed in investigations of heme proteins, photosynthetic systems and tryptophan-containing proteins.

Common dynamics of globin family proteins.

The recently discovered new members of the globin family, neurogobin and cytoglobin, are the object of sustained structural and functional studies aimed at understanding their physiological role and elucidating the impact of their bis‐his heme hexacoordination. However, no studies have yet considered the dynamics of this protein family, an essential link between structure and function. In this communication, we present normal mode analysis results for neuroglobin, cytoglobin, hemoglobin and myoglobin to provide exploratory insights into globin characteristic motions. Our results show a clear correlation in the protein dynamics of this family. All four globins exhibit a high degree of correlated displacements involving residues in the C, E and F helices and link regions. They suggest that these motions play an important role in the reversible oxygen binding function of these proteins. Further, our results may help rationalize some functional features of the 6c‐globins in that they alone exhibit correlated displacements of the G‐helix region.

Rapid procedure for the isolation of cytochrome c peroxidase

Abstract A simple, rapid method is described for the preparation of cytochrome c peroxidase from bakers yeast. The procedure involves lysis of the yeast in the presence of ethyl acetate, extraction of the peroxidase in 0.05 M sodium acetate buffer, pH 5.0, and the concentration of the crude extract on a DEAE-agarose column. The DEAE eluate is further concentrated by ultrafiltration, and gel filtration of the concentrate results in a highly purified form of the enzyme. Consistent yields with 80% recovery are easily obtained. Protein isolated by this method in the presence or absence of the protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), contains purely high-spin Fe(III) heme as monitored by its resonance Raman spectrum.

Mutational analysis of allosteric activation and inhibition of glucokinase.

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.

The importance of vibronic perturbations in ferrocytochrome c spectra: A reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations

We have measured and analyzed the low-temperature (T=10 K) absorption spectrum of reduced horse heart and yeast cytochrome c. Both spectra show split and asymmetric Q(0) and Q(upsilon) bands. The spectra were first decomposed into the individual split vibronic sidebands assignable to B(1g) (nu15) and A(2g) (nu19, nu21, and nu22) Herzberg-Teller active modes due to their strong intensity in resonance Raman spectra acquired with Q(0) and Q(upsilon) excitations. The measured band splittings and asymmetries cannot be rationalized solely in terms of electronic perturbations of the heme macrocycle. On the contrary, they clearly point to the importance of considering not only electronic perturbations but vibronic perturbations as well. The former are most likely due to the heterogeneity of the electric field produced by charged side chains in the protein environment, whereas the latter reflect a perturbation potential due to multiple heme-protein interactions, which deform the heme structure in the ground and excited states. Additional information about vibronic perturbations and the associated ground-state deformations are inferred from the depolarization ratios of resonance Raman bands. The results of our analysis indicate that the heme group in yeast cytochrome c is more nonplanar and more distorted along a B(2g) coordinate than in horse heart cytochrome c. This conclusion is supported by normal structural decomposition calculations performed on the heme extracted from molecular-dynamic simulations of the two investigated proteins. Interestingly, the latter are somewhat different from the respective deformations obtained from the x-ray structures.

Metal coordination influences substrate binding in horseradish peroxidase

Abstract To clarify the role of metal ion coordination in horseradish peroxidase C (HRPC), the effect of pressure and of an externally applied electric field on spectral holes was compared for both metal-free and Mg-mesoporphyrin-substituted horseradish peroxidase C (MP-HRP and MgMP-HRP), as affected by the binding of 2-naphthohydroxamic acid (NHA). The data are compared to earlier studies performed on the same derivatives. Results obtained for MP-HRP show the presence of a predominant MP tautomer, as well as that of another small population with different pocket field and isothermal compressibility (0.12 vs 0.24 GPa−1). Binding NHA induces the formation of two new almost equal populations of MP-HRP tautomer complexes and the protein compressibility in both forms is increased to 0.50 and 0.36 GPa−1. The protein structure becomes much softer than in the absence of NHA. Binding the same substrate to MgMP-HRP resulted in MgMP adopting a single conformation with no compressibility changes, while without NHA, two forms were possible. Stark effect results show charge rearrangement upon substrate binding in both cases. We propose that it is the presence of the metal that stabilizes the structure during the reorganization of the protein matrix induced by the substrate binding event. With the metal, only one conformation is adopted, without significant structural rearrangement but with charge redistribution. The dissociation constants determined for NHA binding to both derivatives and to native HRPC show that studies using mesoporphyrin and Mg-mesoporphyrin derivatives are relevant to investigating the specificity of the substrate-binding pocket in this enzyme.