Identifying sequence determinants of reduction potentials of metalloproteins

The reduction potential of an electron transfer protein is one of its most important functional characteristics. Although the type of redox site and the protein fold are the major determinants of the reduction potential of a redox-active protein, its amino acid sequence may tune the reduction potential as well. Thus, homologous proteins can often be divided into different classes, with each class characterized by a biological function and a reduction potential. Site-specific mutagenesis of the sequence determinants of the differences in the reduction potential between classes should change the reduction potential of a protein in one class to that of the other class. Here, a procedure is presented that combines energetic and bioinformatic analysis of homologous proteins to identify sequence determinants that are also good candidates for site-specific mutations, using the [4Fe–4S] ferredoxins and the [4Fe–4S] high-potential iron–sulfur proteins as examples. This procedure is designed to guide site-specific mutations or more computationally expensive studies, such as molecular dynamics simulations. To make the procedure more accessible to the general scientific community, it is being implemented into CHARMMing, a Web-based portal, with a library of density functional theory results for the redox site that are used in the setting up of Poisson–Boltzmann continuum electrostatics calculations for the protein energetics.     

Redox potentials of some metalloproteins]

The standard redox potentials of soluble cytochromes c isolated from the green alga Chlorella and the blue-green algae Spirulina and Aphanezomenon were determined by potentiometric titration and found to be equal to +380 mB, +330 mB and +357 AB, respectively. The standard redox potentials of plastocyanin preparations from Pisum sativum and Atriplex leaves were also determined and found close to those of soluble cytochromes c, i. e. +395 mB and +375 mB, respectively. The metalloproteins studied were shown to belong to monoelectron carriers operating at the donor sites of photosystem I.     

Paramagnetic effects in NMR for protein structures and ensembles: Studies of metalloproteins

Paramagnetic effects on the NMR spectra are known to encode information on structure, electronic properties and dynamics hardly accessible with any other technique, especially in the field of biological systems. Paramagnetism-based restraints are conveniently used for the de novo determination of protein structures, the structural refinement starting from crystallographic models, and for the determination of the internal arrangement of domains with known structures. Conformational variability can also be profitably interrogated including the possibility of uncovering the presence of states with very low population. The recent advances in the quantum chemistry treatment of paramagnetic NMR effects has provided new momentum to the field, allowing for the refinement of protein structures at the metal coordination site to an unprecedented resolution.     

Influence of the protein environment on the redox potentials of flavodoxins from Clostridium beijerinckii

The flavin mononucleotide (FMN) quinones in flavodoxin have two characteristic redox potentials, namely, Em(FMNH./FMNH-) for the one-electron reduction of the protonated FMN (E1) and Em(FMN/FMNH.) for the proton-coupled one-electron reduction (E2). These redox potentials in native and mutant flavodoxins obtained from Clostridium beijerinckii were calculated by considering the protonation states of all titratable sites as well as the energy contributed at the pKa value of FMN during protonation at the N5 nitrogen (pKa(N5)). E1 is sensitive to the subtle differences in the protein environments in the proximity of FMN. The protein dielectric volume that prevents the solvation of charged FMN quinones is responsible for the downshift of 130-160 mV of the E1 values with respect to that in an aqueous solution. The influence of the negatively charged 5'-phosphate group of FMN quinone on E1 could result in a maximum shift of 90 mV. A dramatic difference of 130 mV in the calculated E2 values of FMN quinone of the native and G57T mutant flavodoxins is due to the difference in the pKa(N5) values. This is due to the difference in the influence exerted by the carbonyl group of the protein backbone at residue 57.     

Characterizing the effects of d-amphetamine on temporal discrimination

Although the effects of dopamine agonists on temporal discrimination have been widely studied, conclusions as to their behavioral and neurophysiological effects are difficult due to a number of discrepant findings in the literature. This study examined whether a previously unexplored procedural difference could account for some of these discrepancies. In three experiments, pigeons categorized the duration of temporal samples during two variants of the interval-bisection procedure. In the position variant, responses to a side key that corresponded to the sample duration produced food. In the color-matching variant, responses to the key color that corresponded to the sample duration produced food. Across experiments, d-amphetamine produced a general disruption of temporal discrimination not accompanied by over or underestimation of time in both procedures. This effect occurred whether pigeons were exposed to both procedures within session (Experiment 1) or across conditions (Experiments 2 and 3) and whether the pigeons were drug experienced (Experiments 1 and 2) or naïve (Experiment 3). Analyses of the cumulative normal functions fitted to the proportion-long response data indicated that d-amphetamine produced its effects by selectively decreasing stimulus control, rather than by affecting timing.     

Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwalds approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E° values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.     

Importance of polarization effect in the study of metalloproteins: Application of polarized protein specific charge scheme in predicting the reduction potential of azurin

Molecular dynamics (MD) simulation is commonly used in the study of protein dynamics, and in recent years, the extension of MD simulation to the study of metalloproteins is gaining much interest. Choice of force field is crucial in MD studies, and the inclusion of metal centers complicates the process of accurately describing the electrostatic environment that surrounds the redox centre. Herein, we would like to explore the importance of including electrostatic contribution from both protein and solvent in the study of metalloproteins. MD simulations with the implementation of thermodynamic integration will be conducted to model the reduction process of azurin from Pseudomonas aeruginosa. Three charge schemes will be used to derive the partial charges of azurin. These charge schemes differ in terms of the amount of immediate environment, respective to copper, considered during charge fitting, which ranges from the inclusion of copper and residues in the first coordination sphere during density functional theory charge fitting to the comprehensive inclusion of protein and solvent effect surrounding the metal centre using polarized protein‐specific charge scheme. From the simulations conducted, the relative reduction potential of the mutated azurins respective to that of wild‐type azurin (ΔE_cal) were calculated and compared with experimental values. The ΔE_cal approached experimental value with increasing consideration of environmental effect hence substantiating the importance of polarization effect in the study of metalloproteins. This study also attests the practicality of polarized protein‐specific charge as a computational tool capable of incorporating both protein environment and solvent effect into MD simulations. Proteins 2014; 82:2209–2219. © 2014 Wiley Periodicals, Inc.     

A new method to determine the structure of the metal environment in metalloproteins: investigation of the prion protein octapeptide repeat Cu2+ complex

Since high-intensity synchrotron radiation is available, extended X-ray absorption fine structure spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu²⁺ complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.     

Characterizing the microenvironment surrounding protein sites.

Sites are microenvironments within a biomolecular structure, distinguished by their structural or functional role. A site can be defined by a three-dimensional location and a local neighborhood around this location in which the structure or function exists. We have developed a computer system to facilitate structural analysis (both qualitative and quantitative) of biomolecular sites. Our system automatically examines the spatial distributions of biophysical and biochemical properties, and reports those regions within a site where the distribution of these properties differs significantly from control nonsites. The properties range from simple atom-based characteristics such as charge to polypeptide-based characteristics such as type of secondary structure. Our analysis of sites uses non-sites as controls, providing a baseline for the quantitative assessment of the significance of the features that are uncovered. In this paper, we use radial distributions of properties to study three well-known sites (the binding sites for calcium, the milieu of disulfide bridges, and the serine protease active site). We demonstrate that the system automatically finds many of the previously described features of these sites and augments these features with some new details. In some cases, we cannot confirm the statistical significance of previously reported features. Our results demonstrate that analysis of protein structure is sensitive to assumptions about background distributions, and that these distributions should be considered explicitly during structural analyses.     

Characterizing the microenvironment surrounding phosphorylated protein sites

Protein phosphorylation plays an important role in various cellular processes. Due to its high complexity, the mechanism needs to be further studied. In the last few years, many methods have been contributed to this field, but almost all of them investigated the mechanism based on protein sequences around protein sites. In this study, we implement an exploration by characterizing the microenvironment surrounding phosphorylated protein sites with a modified shell model, and obtain some significant properties by the rank-sum test, such as the lack of some classes of residues, atoms, and secondary structures. Furthermore, we find that the depletion of some properties affects protein phosphorylation remarkably. Our results suggest that it is a meaningful direction to explore the mechanism of protein phosphorylation from microenvironment and we expect further findings along with the increasing size of phosphorylation and protein structure data.     

Side-chain torsional potentials: effect of dipeptide, protein, and solvent environment.

Side-chain torsional potentials in the bovine pancreatic trypsin inhibitor are calculated from empirical energy functions by use of the known X-ray structure of the protein and the rigid-geometry mapping technique. The potentials are analyzed to determine the roles and relative importance of contributions from the dipeptide backbone, the protein, and the crystalline environment of solvent and other protein molecules. The structural characteristics of the side chains determine two major patterns of energy surfaces, E(X1,X2): a gamma-branched pattern and a pattern for longer, straight side chains (Arg, Lys, Glu, and Met). Most of the dipeptide potential curves and surfaces have a local minimum corresponding to the side-chain torsional angles in the X-ray structure. Addition of the protein forces sharpens and/or selects from these minima, providing very good agreement with the experimental conformation for most side chains at the surface or in the core of the protein. Inclusion of the crystalline environment produces still better results, especially for the side chains extending away from the protein. The results are discussed in terms of the details of the interactions due to the surrounding, calculated solvent-accessibility figures and the temperature factors derived from the crystallographic refinement of the pancreatic trypsin inhibitor.     

The one-electron reduction potentials of NAD

The one-electron reduction potential (E¹₇) of NAD⁺ has been determined by pulse radiolysis to be ?0.94 V. E²₇ (E¹₇ for the free radical, NAD^.) is +0.30 V. E¹₇ for 1-methylisonicotinamide is ?0.77 V.     

Influence of ionic environment on the relationship between pre- and postsynaptic potentials

The effects of various ions on the relationship between pre- and postsynaptic potentials were studied using untreated squid giant synapses, or those injected presynaptically with tetraethylammonium ions (TEA) in the presence of 10^?6 g/ml tetrodotoxin (TTX). The synaptic transfer function was, in general, augmented by increasing [Ca²⁺]₀ or by reducing [Mg²⁺]₀. Opposite results were found by lowering either [Na⁺]₀ or [Ca²⁺]₀, or by increasing [K⁺]₀ or [Mg²⁺]₀. When [Ca²⁺]₀ was removed, presynaptically applied depolarizations failed to produce both “On-” and “Off-PSP's.” Electrophoretically injected Ca²⁺ into the presynaptic terminal reduced synaptic transmission. The minimal level of presynaptic membrane potential produced by an applied outward current pulse, which suppressed On-PSP completely, averaged 106 mV inside positive (ranging between 52–205 mV from 12 preparations). The potential level was shifted more negatively on lowering [Ca²⁺]₀ and more positively in high [Ca²⁺]₀-media. However, altering the [Na⁺]₀ did not change the suppression level appreciably. Under these ionic circumstances the maximum amplitude of On-PSP, as well as Off-PSP, was markedly changed, but the level of the presynaptic depolarization required to evoke a maximum On-PSP appeared to be unchanged, and the average value was 50 mV from the resting membrane potential level. Although the data are only qualitative, they appear to support the “Ca hypothesis” for the transmitter release and its shut-off mechanism. Replacement of 423 mM Cl^? by Br^? or with isethionate did not affect synaptic transmission.