β-turn tendency in N-methylated peptides with dehydrophenylalanine residue: DFT study

The tendency to adopt β‐turn conformation by model dipeptides with α,β‐dehydrophenylalanine (ΔPhe) residue in the gas phase and in solution is investigated by theoretical methods. We pay special attention to a dependence of conformational properties on the side‐chain configuration of dehydro residue and the influence of N‐methylation on β‐turn stability. An extensive computational study of the conformational preferences of Z and E isomers of dipeptides Ac‐Gly‐(E/Z)‐ΔPhe‐NHMe ( 1a / 1b ) and Ac‐Gly‐(E/Z)‐ΔPhe‐NMe₂ ( 2a / 2b ) by B3LYP/6‐311++G(d,p) and MP2/6‐311++G(d,p) methods is reported. It is shown that, in agreement with experimental data, Ac‐Gly‐(Z)‐ΔPhe‐NHMe has a great tendency to adopt β‐turn conformation. In the gas phase the type II β‐turn is preferred, whereas in the polar environment, the type I. On the other hand, dehydro residue in Ac‐Gly‐(E)‐ΔPhe‐NHMe has a preference to adopt extended conformations in all environments. N‐methylation of C‐terminal amide group, which prevents the formation of 1←4 intramolecular hydrogen bond, change dramatically the conformational properties of studied dehydropeptides. Especially, the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Ac‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuo and in the polar environment. On the contrary, N‐methylated E isomer (Ac‐Gly‐(E)‐ΔPhe‐NMe₂) can easier adopt β‐turn conformation, but the backbone torsion angles (?₁, ψ₁, ?₂, ψ₂) are off the limits for common β‐turn types. © 2012 Wiley Periodicals, Inc. Biopolymers 97:518–528, 2012.     

Acidic residue modifications restore chaperone activity of β-casein interacting with lysozyme

An important factor in medicine and related industries is the use of chaperones to reduce protein aggregation. Here we show that chaperone ability is induced in β-casein by modification of its acidic residues using Woodward's Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study β-casein chaperone activities. The mechanism for chaperone activity of the modified β-casein was determined using UV-vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoretical calculations. Our results indicated that the β-casein destabilizes the lysozyme and increases its aggregation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of β-casein resulting in its altered net negative charge upon interactions with lysozyme. The reversible stability of lysozyme increased in the presence of WRK-modified β-casein, and hence its aggregation rate decreased. These results demonstrate the enhanced chaperone activity of modified β-casein and its protective effects on lysozyme refolding.     

Amino acid sequences around the cysteine residue of calf lens α-crystallin

1. Calf lens alpha-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.     

Effect of phosphatidylglycerol on conformation and micro-environment of tyrosyl residue in photosystem Ⅱ

The structural aspects in the interaction of phosphatidylglycerol (PG) with photosystem II (PSII),mainly the effect of PG on conformation and microenvironment of tyrosine residues of PSII proteins were studied by Fourier transform infrared (FTIR) spectroscopy.It was found that the binding of PG to PSII particle induces changes in the conformation and micropolarity of phenol ring in the tyrosine residues.In other words,the PG effect on the PSII results in blue shift of the stretch vibrational band in the phenol ring from 1620 to 1500 cm-1 with the enhancement of the absorbance intensity.Additionally,a new spectrum of hydrogen bond was also observed.The results imply that the hydrogen-bond formation between the OH group of phenol and one of PG might cause changes in the structures of tyrosine residues in PSII proteins.     

Presence of an essential arginyl residue in D-β-hydroxybutyrate dehydrogenase from mitochondrial inner membrane

D-β-hydroxybutyrate dehydrogenase, a lipid requiring enzyme, is rapidly and completely inactivated by phenylglyoxal, 2,3-butanedione and 1,2-cyclohexanedione. Inactivation, which occurs at the millimolar range, depends on the nature of buffer, borate ions are required to get enzyme inactivation by 2,3-butanedione. Most of the inactivation follows a pseudo first order kinetics, the stoichiometry being of one to one. Presence of NAD⁺ or methylmalonate (a substrate-like compound) prior addition of inhibitor does not affect inactivation, while methylmalonate in presence of NAD⁺ strongly protects against inactivation. Chemical modification of the enzyme does not affect K_D of NAD while K_M of β-hydroxybutyrate and Ki of methylmalonate (protecting agent) increase. In view of the high specificity of these inhibitors for arginyl residues of proteins, these results are in favour of the presence of at least one arginyl residue essential for enzyme activity and located in, or near the substrate binding site.     

Contribution of each Trp residue toward the intrinsic fluorescence of the Giα1 protein

G_iα1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of G_iα1 bound to GDP•AMF or GTPγS with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the α-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP•AMF or GTPγS, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTPγS, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a π-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic π-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of G_iα1.     

Do residue levels of polychlorinated biphenyls (PCBs) in human blood produce mild hypothyroidism?

PCBs are nearly ubiquitous environmental contaminants, occurring in most human adipose tissue and blood samples. It has recently been recognized that PCBs and related compounds share important structural properties with thyroid hormones and can bind thyroid hormone binding proteins. It is reasonable that such specific binding interactions can modulate the distribution of these compounds in the body and alter hormone-protein interactions that are responsible for the maintenance of normal thyroid status. Most of the available evidence indicates that the levels of free thyroid hormones in plasma are a reflection of the maintenance of normal thyroid status in any individual. A theoretical model for the transport of thyroid hormones in blood has been extended to estimate the modulating effects of PCBs on free thyroid hormones. Using conservative assumptions based on experimental data, our calculations indicate that PCB concentrations normally found in humans can effect significant increases in free thyroxine levels in serum by competing with serum thyroid hormone binding proteins. Experimental data are discussed which support the proposal that antagonist binding of PCBs to thyroid hormone binding proteins in serum could produce varying degrees of hypothyroidism. The biological result is compatible with the "equilibrium hypothesis" in which thyroid hormone redistributes between specific and nonspecific binding proteins rather than emphasizing the importance of free hormone as the active moiety.     

Identification of an essential cysteinyl residue for the structure of glutamine synthetase α from Phaseolus vulgaris

We have studied the possible role, in a plant glutamine synthetase (GS), of the different cysteinyl residues present in this enzyme. For this purpose we carried out the site-directed mutagenesis of the cDNA for α-GS polypeptide from Phaseolus vulgaris in the positions corresponding to Cys-92, Cys-159, and Cys-179, followed by heterologous expression in E. coli and enzymatic characterisation of WT and mutant proteins. The results show that neither Cys-92 nor Cys-179 residues were essential for enzyme activity, but the replacement of Cys-159 by alanine or serine strongly affects the quaternary structure and function of the GS enzyme molecule, resulting in a complete loss of enzymatic activity. Other studies using sulfhydryl specific reagents such as pHMB (p-hydroxymercuribenzoate) or DTNB (5,5′-dithiobis-2-nitrobenzoate) confirmed that the profound inhibition produced is associated with an important alteration of the quaternary structure of GS, and suggest that Cys-159 might be the residue responsible for the enzyme inhibition. All these results suggest that the Cys-159 residue is essential for the enzyme structure. The results are also consistent with previous reports based on classical biochemistry studies indicating the presence of essential cysteinyl residues for the enzyme activity of higher plant GS.     

A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe–4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe–4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe–4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe–4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (> 100 mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.     

The influence of solvent on conformational properties of peptides with Aib residue—a DFT study

The conformational propensities of the Aib residue on the example of two model peptides Ac-Aib-NHMe (1) and Ac-Aib-NMe₂ (2), were studied by B3LYP and M06-2X functionals, in the gas phase and in the polar solvents. To verify the reliability of selected functionals, we also performed MP2 calculations for the tested molecules in vacuum. Polarizable continuum models (PCM and SMD) were used to estimate the solvent effect. Ramachandran maps were calculated to find all energy minima. Noncovalent intramolecular interactions due to hydrogen-bonds and dipole attractions between carbonyl groups are responsible for the relative stabilities of the conformers. In order to verify the theoretical results, the available conformations of similar X-ray structures from the Cambridge Crystallographic Data Center (CCDC) were analyzed. The results of the calculations show that both derivatives with the Aib residue in the gas phase prefer structures stabilized by intramolecular N–H?O hydrogen bonds, i.e., C₅ and C₇ conformations, while polar solvent promotes helical conformation with φ, ψ values equal to +/?60°, +/?40°. In addition, in the case of molecule 2, the helical conformation is the only one available in the polar environment. This result is fully consistent with the X-ray data.

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Graphical abstract Effect of solvent on the Ramachandran maps of the model peptides with Aib residue

     

Mutation of tyrosine residue 857 in the PDGF β-receptor affects cell proliferation but not migration

Activation of platelet-derived growth factor (PDGF) receptors occurs through ligand-induced dimerization and autophosphorylation. In this study, we investigated the effects of mutation of tyrosine residue 857 (Y857) in the activation loop of the PDGF β-receptor (PDGFRβ) to phenylalanine (Y857F). In agreement with previous observations, we found that PDGFRβ^Y857F had a severely diminished in vitro kinase activity. However, in vivo the overall amount of tyrosine phosphorylation of PDGFRβ^Y857F was similar to that of the wild-type receptor, except for the tyrosine residue 771 (Y771) which displayed a stronger phosphorylation in the mutant receptor. Analysis of the ability to induce signal transduction revealed that the PDGFRβ^Y857F mutant had an attenuated activation of Akt and Erk1/2 MAP kinase. In contrast, the mutant receptor efficiently mediated phosphorylation of the ubiquitin-ligase c-Cbl that participates in receptor internalization and degradation, and PLCγ which has previously been shown to be connected with various cellular responses, including migration. However, the protein tyrosine phosphatase SHP-2, implicated in the PDGF-induced mitogenic response, together with the adaptor proteins Alix and Stam, involved in intracellular sorting of receptor, was not phosphorylated in cells expressing PDGFRβ^Y857F. We found that both receptor variants were internalized from the cell surface and degraded at a comparable rate. Interestingly, PDGFRβ^Y857F was unable to mediate PDGF-BB-induced mitogenic signaling, whereas it could elicit a chemotactic response.     

A critical histidine residue within LIMP-2 mediates pH sensitive binding to its ligand β-glucocerebrosidase

The lysosomal membrane protein type 2 is a novel identified lysosomal sorting receptor for β-glucocerebrosidase (GC). Mutations in both genes underlie human pathologies causing action myoclonus-renal failure syndrome (AMRF) and Gaucher disease (GD), respectively. We now demonstrate that the lumenal acidification mediated by the vacuolar (H(+) )-ATPase triggers the dissociation of LIMP-2 and GC in late endosomal/lysosomal compartments. Moreover, we identified a single histidine residue in LIMP-2 that is necessary for LIMP-2 and GC binding. This residue is in close proximity to a proposed coiled-coil domain, which determines the binding to GC and may function as a critical pH sensor.     

Role of an isoform-specific serine residue in FMN–heme electron transfer in inducible nitric oxide synthase

In the crystal structure of a calmodulin (CaM)-bound FMN domain of human inducible nitric oxide synthase (NOS), the CaM-binding region together with CaM forms a hinge, and pivots on an R536(NOS)/E47(CaM) pair (Xia et al. J Biol Chem 284:30708-30717, 2009). Notably, isoform-specific human inducible NOS S562 and C563 residues form hydrogen bonds with the R536 residue through their backbone oxygens. In this study, we investigated the roles of the S562 and C563 residues in the NOS FMN-heme interdomain electron transfer (IET), the rates of which can be used to probe the interdomain FMN/heme alignment. Human inducible NOS S562K and C563R mutants of an oxygenase/FMN (oxyFMN) construct were made by introducing charged residues at these sites as found in human neuronal NOS and endothelial NOS isoforms, respectively. The IET rate constant of the S562K mutant is notably decreased by one third, and its flavin fluorescence intensity per micromole per liter is diminished by approximately 24?%. These results suggest that a positive charge at position 562 destabilizes the hydrogen-bond-mediated NOS/CaM alignment, resulting in slower FMN-heme IET in the mutant. On the other hand, the IET rate constant of the C563R mutant is similar to that of the wild-type, indicating that the mutational effect is site-specific. Moreover, the human inducible NOS oxyFMN R536E mutant was constructed to disrupt the bridging CaM/NOS interaction, and its FMN-heme IET rate was decreased by 96?%. These results demonstrated a new role of the isoform-specific serine residue of the key CaM/FMN(NOS) bridging site in regulating the FMN-heme IET (possibly by tuning the alignment of the FMN and heme domains).     

Quantification of cyanuric acid residue in human urine using high performance liquid chromatography–tandem mass spectrometry

Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography–tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00–200 ng/mL. The limit of detection was 0.60 ng/mL and the relative standard deviations were 2.8–10.5% across the calibration range. The relative recovery of cyanuric acid was 100–104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.     

Synthesis of a novel glycosaminoglycan pentasaccharide serine having anN-acetylgalactosamine residueα-linked to the core linkage tetrasaccharide

A novel pentaosyl serine; GalNAc(1–4)GlcA(1–3)Gal(1–3)Gal(1–4)Xyl(1–3)Ser (2), a putative intermediate of chondroitin sulfate and/or heparan sulfate biosynthesis, was synthesized.     

Effects of tryptophan residue fluorination on streptavidin stability and biotin–streptavidin interactions via molecular dynamics simulations

Due to its highly specific and very strong binding, the (strept)avidin–biotin system forms the basis for numerous applications in the life sciences: immunoassays, DNA detection systems, affinity chromatography, etc. Fine-tuning of the ligand binding abilities of this system might provide new technologies with relevance to nanoscale research. Here, we report our computational investigations on wild type (WT) and modified streptavidin (SAV), assessing the impact of fluorination of tryptophan residues on biotin binding ability. Complexes of biotin with four SAV protein variants (WT-SAV, 4fW-SAV, 5fW-SAV and 6fW-SAV) were studied. We found that protein stability and folding are predicted to be weakly affected by fluorination. The host protein binding pocket decreases its ability to form numerous hydrogen bonds to biotin in the case of the 4fW-SAV variant. Conversely, the 5fW-SAV mutant is predicted to have an even more stable ligand–host hydrogen bonding network than WT-SAV. Thermodynamic perturbation investigations predict a decrease in biotin binding free energy from 3.0 to 6.5 kcal/mol per tetrameric host, with the 5fW-SAV mutant being least affected. Overall, the computational findings indicate that 6fW-SAV and, especially, 5fW-SAV to be promising variants of streptavidin for potential modifiable picomolar binding of the biotin ligand family. Figure Hydrogen bonding framework of the biotin–streptavidin system Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Is the glutamate residue Glu-373 the proton acceptor of the excitatory amino acid carrier 1?

Glutamate transport by the neuronal excitatory amino acid carrier (EAAC1) is accompanied by the coupled movement of one proton across the membrane. We have demonstrated previously that the cotransported proton binds to the carrier in the absence of glutamate and, thus, modulates the EAAC1 affinity for glutamate. Here, we used site-directed mutagenesis together with a rapid kinetic technique that allows one to generate sub-millisecond glutamate concentration jumps to locate possible binding sites of the glutamate transporter for the cotransported proton. One candidate for this binding site, the highly conserved glutamic acid residue Glu-373 of EAAC1, was mutated to glutamine. Our results demonstrate that the mutant transporter does not catalyze net transport of glutamate, whereas Na(+)/glutamate homoexchange is unimpaired. Furthermore, the voltage dependence of the rates of Na(+) binding and glutamate translocation are unchanged compared with the wild-type. In contrast to the wild-type, however, homoexchange of the E373Q transporter is completely pH-independent. In line with these findings the transport kinetics of the mutant EAAC1 show no deuterium isotope effect. Thus, we suggest a new transport mechanism, in which Glu-373 forms part of the binding site of EAAC1 for the cotransported proton. In this model, protonation of Glu-373 is required for Na(+)/glutamate translocation, whereas the relocation of the carrier is only possible when Glu-373 is negatively charged. Interestingly, the Glu-373-homologous amino acid residue is glutamine in the related neutral amino acid transporter alanine-serine-cysteine transporter. The function of alanine-serine-cysteine transporter is neither potassium- nor proton-dependent. Consequently, our results emphasize the general importance of glutamate and aspartate residues for proton transport across membranes.     

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K_d) and the net binding energy from the agonist for gating (ΔG_B) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K_d and essentially no change in ΔG_B. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K_d but no change in ΔG_B. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.