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PubMed2002

Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity.

The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.

Amino Acid Sequence↗
PubMed2002

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects.

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.

Amino Acid Sequence↗
PubMed2002

Structural and thermodynamic studies on a salt-bridge triad in the NADP-binding domain of glutamate dehydrogenase from Thermotoga maritima: cooperativity and electrostatic contribution to stability.

Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability. While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles. In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193. The X-ray structure of the domain was solved at 1.43 A and reveals the salt-bridge network with surrounding solvent molecules in detail. All three participating charges in the network were mutated to alanine in all combinations. The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein. Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue. Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea. Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol. The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles.

Alanine↗
PubMed2002

Electrostatic interactions in ubiquitin: stabilization of carboxylates by lysine amino groups.

To explore electrostatic interactions in ubiquitin, pK(a) values have been determined by NMR for all 12 carboxyl groups in wild-type ubiquitin and in variants where single lysines have been replaced by neutral residues. Aspartate pK(a) values in ubiquitin range from 3.1 to 3.8 and are generally less than model compound values. Most aspartate pK(a) values are within 0.2 pH unit of those predicted with a simple Tanford-Kirkwood model. Glutamate pK(a) values range from 3.8 to 4.5, close to model compound values and differing by 0.1-0.8 pH unit from calculated values. To determine the role of positive charges in modulating carboxyl pK(a) values, we mutated lysines at positions 11, 29, and 33 to glutamine and threonine. NMR studies with these six single-site mutants reveal significant interactions of Lys 11 and Lys 29 with Glu 34 and Asp 21, respectively: pK(a) values for Glu 34 and Asp 21 increase by approximately 0.5-0.8 pH unit, similar to predicted values, when the lysines are replaced by neutral residues. In contrast, the predicted interaction between Lys 33 and Glu 34 is not observed experimentally. In some instances, substitution of lysine by glutamine and threonine did not lead to the same changes in carboxyl pK(a) values. These may reflect new short-range interactions between the mutated residues and the carboxyl groups. Carboxyl pK(a) shifts > 0.5 pH unit result from mutations at groups that are <5 A from the carboxyl group. No interactions are observed at >10 A.

Amides↗
PubMed2002

Characterization of large peptide fragments derived from the N-terminal domain of the ribosomal protein L9: definition of the minimum folding motif and characterization of local electrostatic interactions.

A set of peptides derived from the N-terminal domain of the ribosomal protein L9 (NTL9) have been characterized in an effort to define the minimum unit of this domain required to fold and to provide model peptides for the analysis of electrostatic interactions in the unfolded state. NTL9 is a 56-residue alpha-beta protein with a beta1-loop-beta2-alpha1-beta3-alpha2 topology. The beta-sheet together with the first helix comprise a simple example of a common supersecondary motif called the split beta-alpha-beta fold. Peptides corresponding to the beta1-loop-beta2 unit are unstructured even when constrained by an introduced disulfide. The pK(a)s of Asp-8 and Glu-17 in these peptides are slightly lower than the values found for shorter peptides but are considerably higher than the values in NTL9. A 34-residue peptide, which represents the beta1-loop-beta2-alpha1 portion of NTL9, is also unstructured. In contrast, a 39-residue peptide corresponding to the entire split beta-alpha-beta motif is folded and monomeric as judged by near- and far-UV CD, two-dimensional NMR, ANS binding experiments, pK(a) measurements, and analytical ultracentrifugation. The fold is very similar to the structure of this region in the intact protein. Thermal and urea unfolding experiments show that it is cooperatively folded with a DeltaG degrees of unfolding of 1.8-2.0 kcal/mol and a T(m) of 58 degrees C. This peptide represents the first demonstration of the independent folding of an isolated split beta-alpha-beta motif, and is one of only four naturally occurring sequences of fewer than 40 residues that has been shown to fold cooperatively in the absence of disulfides or ligand binding.

Amino Acid Motifs↗
PubMed2004

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover.

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of p-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site-to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of p-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2003

Electrostatic tuning of ion conductance in potassium channels.

Members of the K(+) channel family display remarkable conservation of sequence and structure of the ion selectivity filter, whereas the rates of K(+) turnover vary widely within the family. Here we show that channel conductance is strongly influenced by charge at the channel's intracellular mouth. Introduction of a ring of negative charges at this position in KcsA, a bacterial K(+) channel, augments the conductance in a pH-dependent manner. These results are explained by a simple electrostatic effect based on known channel structures, where the negative charges serve to alter the electrical potential at the inner mouth and, thus, to increase the local K(+) concentration. In addition, removal of the conserved negative charges at equivalent positions in a high-conductance eukaryotic K(+) channel leads to a decrease in conductance.

Amino Acid Sequence↗
PubMed2004

Role of protein-water interactions and electrostatics in alpha-synuclein fibril formation.

Deposition of misfolded alpha-synuclein is a critical factor in several neurodegenerative disorders. Filamentous alpha-synuclein is the major component of Lewy bodies and Lewy neurites, the intracellular inclusions in the dopaminergic neurons of the substantia nigra, which are considered the pathological hallmark of Parkinson's disease. We show here that anions induce partial folding of alpha-synuclein at neutral pH, forming a critical amyloidogenic intermediate, which leads to significant acceleration of the rate of fibrillation. The magnitude of the accelerating effect generally followed the position of the anions in the Hofmeister series, indicating a major role of protein-water-anion interactions in the process at salt concentrations above 10 mM. Below this concentration, electrostatic effects dominated in the mechanism of anion-induced fibrillation. The acceleration of fibrillation by anions was also dependent on the cation. Moderate concentrations of anions affected both the rates of nucleation and the elongation of alpha-synuclein fibrillation, primarily via their effect on the interaction of the protein with water.

Acids↗
PubMed2004

Electrostatic contribution of serine phosphorylation to the Drosophila SLBP--histone mRNA complex.

Unlike all other metazoan mRNAs, mRNAs encoding the replication-dependent histones are not polyadenylated but end in a unique 26 nucleotide stem-loop structure. The protein that binds the 3' end of histone mRNA, the stem-loop binding protein (SLBP), is essential for histone pre-mRNA processing, mRNA translation, and mRNA degradation. Using biochemical, biophysical, and nuclear magnetic resonance (NMR) experiments, we report the first structural insight into the mechanism of SLBP-RNA recognition. In the absence of RNA, phosphorylated and unphosphorylated forms of the RNA binding and processing domain (RPD) of Drosophila SLBP (dSLBP) possess helical secondary structure but no well-defined tertiary fold. Drosophila SLBP is phosphorylated at four out of five potential serine or threonine sites in the sequence DTAKDSNSDSDSD at the extreme C-terminus, and phosphorylation at these sites is necessary for histone pre-mRNA processing. Here, we provide NMR evidence for serine phosphorylation of the C-terminus using (31)P direct-detect experiments and show that both serine phosphorylation and RNA binding are necessary for proper folding of the RPD. The electrostatic effect of protein phosphorylation can be partially mimicked by a mutant form of SLBP wherein four C-terminal serines are replaced with glutamic acids. Hence, both RNA binding and protein phosphorylation are necessary for stabilization of the SLBP RPD.

Amino Acid Sequence↗
PubMed2005

Electrostatic interaction of a K+ channel RCK domain with charged membrane surfaces.

In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains. To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general functional behavior and identifying purification conditions. Standard detergent solubilization of recombinantly expressed MjK2 required the addition of a high NaCl concentration to the extraction buffer for MjK2 solubilization. The cytoplasmic RCK domain was identified as the region of MjK2 responsible for the dependence of solubilization on high salt concentrations since expression of an MjK2 construct lacking the transmembrane domain, MjK2cd, also required high salt concentrations for extraction from Escherichia coli lipids, a necessary step in the purification of both MjK2 and MjK2cd. MjK2 expression was able to weakly recover growth of K(+) uptake deficient LB2003 cells at 10 mM KCl, suggesting that the channel can conduct K(+) but has a low open probability. Purified MjK2 reconstituted in liposomes generated only limited Rb(+) uptake, blocked by BaCl(2). MjK2cd exhibited direct binding to PC/PS lipid vesicles with a molar partition coefficient (K(1)) of approximately 10(3) M(-)(1), which decreased with both an increase in the salt concentration and a decrease in the anionic lipid ratio. Lipid blot assays revealed that MjK2cd binds most strongly to lipids of increasingly negative charge. These results support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.

Amino Acid Sequence↗
PubMed2005

Chemically modified DNA substrates implicate the importance of electrostatic interactions for DNA unwinding by Dda helicase.

Helicase-catalyzed disruption of double-stranded nucleic acid is vital to DNA replication, recombination, and repair in all forms of life. The relative influence of specific chemical interactions between helicase and the substrate over a series of multistep catalytic events is still being defined. To this end, three modified DNA oligonucleotides were designed to serve as substrates for the bacteriophage T4 helicase, Dda. A 5'-DNA-PNA-DNA-3' chimera was synthesized, thereby, conferring both a loss of charge and altering the conformational flexibility of the oligonucleotide. The second modified oligonucleotide possessed a single methylphosphonate replacement on the phosphate backbone, creating a gap in the charge distribution of the substrate. The third modification introduced an abasic site into the oligonucleotide sequence. This abasic site retains the charge distribution of the normal DNA substrate yet alters the conformational flexibility of the oligonucleotide. The loss of a base also serves to disrupt the hydrogen-bonding lattice, the intramolecular base-stacking interactions, as well as the intermolecular base-stacking interactions between aromatic amino acid side chains and the substrate. Our results indicate that a gap in the charge distribution along the backbone of the substrate has a more pronounced effect upon helicase-catalyzed unwinding than does the loss of a single base. While all three substrates exhibited some degree of inhibition, analysis of both pre-steady-state and excess enzyme experiments places a greater value upon the electrostatic interactions between helicase and the substrate.

Bacteriophage T4↗
PubMed2004

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions.

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.

Amino Acid Sequence↗
PubMed2004

Electrostatic control of the tryptophan radical in cytochrome c peroxidase.

Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.

Crystallography, X-Ray↗
PubMed2005

Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential.

The cell-penetrating peptide (CPP) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. Two possible mechanisms have been proposed to explain the entrance of other CPPs in cells, endosomal-dependent and independent types. In this work, we evaluated the molecular mechanisms of pep-1-mediated cellular uptake of beta-galactosidase (beta-Gal) from Escherichia coli in large unilamellar vesicles (LUV) and HeLa cells. Fluorescence spectroscopy was used to evaluate the translocation process in model systems (LUV). Immunofluorescence microscopy was used to study the translocation in HeLa cells. Enzymatic activity detection enabled us to monitor the internalization of beta-Gal into LUV and the functionality of the protein in the interior of HeLa cells. Beta-Gal translocated into LUV in a transmembrane potential-dependent manner. Likewise, the extent of beta-Gal incorporation was extensively decreased in depolarized cells. Furthermore, beta-Gal uptake efficiency and kinetics were temperature-independent, and beta-Gal did not colocalize with endosomes, lysosomes, or caveosomes. Therefore, beta-Gal translocation was not associated with the endosomal pathway. Although an excess of pep-1 was mandatory for beta-Gal translocation in vivo, transmembrane pores were not formed as concluded from the trypan blue exclusion method. These results altogether indicated that protein uptake both in vitro with LUV and in vivo with HeLa cells was mainly, if not solely, dependent on negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between pep-1 (positively charged) and membranes (negatively charged).

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2005

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D.

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).

Amino Acid Sequence↗
PubMed2006

Intersubunit electrostatic complementarity in the RecA nucleoprotein filament regulates nucleotide substrate specificity and conformational activation.

The Escherichia coli RecA protein is the prototypical member of a family of molecular motors that transduces ATP binding and hydrolysis for mechanical function. While many general mechanistic features of RecA action are known, specific structural and functional insights into the molecular basis of RecA activation remain elusive. Toward a more complete understanding of the interdependence between ATP and DNA binding by RecA, we report the characterization of a mutant RecA protein wherein the aspartate residue at position 100 within the ATP binding site is replaced by arginine. Physiologically, D100R RecA was characterized by an inducible, albeit reduced, activation of the SOS response and a diminished ability to promote cellular survival after UV irradiation. Biochemically, the D100R substitution caused a surprisingly modest perturbation of RecA-ATP interactions and an unexpected and significant decrease in the affinity of RecA for ssDNA. Moreover, in vitro assays of RecA activities requiring the coordinated processing of ATP and DNA revealed (1) a 2-5-fold decrease in steady-state turnover of ATP; (2) no formation of mixed nucleoprotein filaments when wild-type and D100R RecA compete for limiting ssDNA; and (3) no formation of strand exchange reaction products. Taken together, these results suggest that the D100R mutational effects on isolated RecA activities combine synergistically to perturb its higher-order functions. We conclude that the replacement of Asp100 resulted in a change in the electrostatic complementarity between RecA monomers during active filament assembly that prevents the protein from fully accessing the active multimeric state.

Adenosine Diphosphate↗
PubMed2006

Catalysis in glycine N-methyltransferase: testing the electrostatic stabilization and compression hypothesis.

Glycine N-methyltransferase (GNMT) is an S-adenosyl-l-methionine dependent enzyme that catalyzes glycine transformation to sarcosine. Here, we present a hybrid quantum mechanics/molecular mechanics (QM/MM) computational study of the reaction compared to the counterpart process in water. The process takes place through an SN2 mechanism in both media with a transition state in which the transferring methyl group is placed in between the donor (SAM) and the acceptor (the amine group of glycine). Comparative analysis of structural, electrostatic, and electronic characteristics of the in-solution and enzymatic transition states allows us to get a deeper insight into the origins of the enzyme's catalytic power. We found that the enzyme is able to stabilize the substrate in its more active basic form by means of a positively charged residue (Arg175) placed in the active site. However, the maximum stabilization is attained for the transition state. In this case, the enzyme is able to form stronger hydrogen bonds with the positively charged amine group. Finally, we show that in agreement with previous computational studies on other methyltransferases, there is no computational evidence for the compression hypothesis, as was formulated by Schowen (Hegazi, M. F., Borchardt, R. T., and Schowen, R. L. (1979) J. Am. Chem. Soc. 101, 4359-4365).

Catalysis↗
PubMed1996

Characterization of electrostatic and nonelectrostatic components of protein--membrane binding interactions.

A general method was developed to determine the thermodynamic parameters for the interaction of protein with membranes. Protein intrinsic tryptophan fluorescence was quenched by titration with large unilamellar vesicles containing 9,10-dibrominated distearoylphosphatidylcholine (Br4-DSPC) or a small amount of trinitrophenylphosphatidylethanolamine (TNP-PE), Binding was modeled as a bimolecular reaction of free protein with a unit of "n" lipid molecules and a dissociation constant, Kd. The contribution of residual fluorescence and light scattering could be eliminated by using the second derivative of the titration function as the basis for calculations. For the binding of C-terminal channel domain polypeptides(178-190 residues) of the colicin El ion channel, n=50-60 and Kd=2-3 nM at pH 4, ionic strength, I=0.12 M, and anionic lipid content = 40% (surface potential, psi o =-30 mV), conditions for which the protein has high activity. Values of n = 95 and 210 for the binding of a C-terminal 293-residue colicin fragment and the 522 residue intact colicin E1 molecule scale qualitatively according to the increase in molecular size. General methods are presented to distinguish the electrostatic (delta G el) and nonelectrostatic (delta G nel) components of the total delta G for binding. Using Br4DSPC as the quencher, the binding of the channel polypeptide, P178, was characterized by delta G approximately -9.8 kcal/mol, and delta G el approximately -7.0 kcal/mol, and delta G el= -2.8 kcal/mol (psi o = -30 mV). Using TNP-PE as the quencher, similar values of delta G approximately -9.3 to -9.9 kcal/mol were determined, a somewhat smaller value for delta G nel approximately -5.0 kcal/mol, and a correspondingly larger value for deltaGnel approximately -4.9 kcal/mol. The existence of a delta G nel component of this magnitude may distinguish proteins that have the potential to insert into the membrane.

Colicins↗