Application of the two-dimensional nmr techniques on a des(Ala,Gly)-somatostatin analog

Two-dimensional nmr techniques have been carried out for the peak assignment of the spectrum of a somatostatin analog. Two-dimensional J-resolved spectroscopy simplified the rather broad and complicated spectrum to show the center of chemical shifts of each resonance and gave information on the coupling profiles. Another technique, two-dimensional spin-echo correlated spectroscopy, revealed the connectivities between protons which are correlated by weak spin–spin couplings. The combination of the results of these two complementary techniques made it possible for us to assign almost all peaks of the spectrum of the 11-residue somatostatin analog.     

Empirical correlation for the replacement of Ala by Gly: Importance of amino acid secondary intrinsic propensities

A series of Ala vs. Gly mutations at different helical and nonhelical positions of the chemotactic protein CheY, from E. coli, has been made. We have used this information to fit a general analytical equation that describes the free energy changes of an Ala to Gly mutation within ±0.45 kcal mol^?1 with 95% confidence. The equation includes three terms: (1) the change in solvent-accessible hydrophobic surface area, corrected for the possible closure of the cavity left by deleting the C_β of the Ala; (2) the change in hydrophilic area of the nonintramolecularly hydrogen-bonded groups; and (3) the dihedral angles of the position being mutated. This last term extends the calculation to any conformation, not only α-helices. The general applicability of the equation for Ala vs. Gly mutations, when Ala or a small solvent-exposed polar residue is the wild-type residue, has been tested using data from other proteins: barnase, CI2 trypsin inhibitor, T4 lysozyme, and Staphylococcus nuclease. The predictive power of this simple approach offers the possibility of extending it to more complex mutations. © 1995 Wiley-Liss, Inc.     

Substitution of Gly with Ala enhanced the melanoma uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to determine the melanoma targeting property of (99m)Tc-RAD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice and compare with (99m)Tc-RGD-Lys-(Arg(11))CCMSH we previously reported. (99m)Tc-RAD-Lys-(Arg(11))CCMSH exhibited rapid and high tumor uptake (19.91±4.02% ID/g at 2h post-injection) in B16/F1 melanoma-bearing C57 mice. The tumor uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH was 1.51, 1.34 and 1.43 times the tumor uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH at 0.5, 2 and 4h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2h post-injection using (99m)Tc-RAD-Lys-(Arg(11))CCMSH as an imaging probe. The substitution of Gly with Ala significantly enhanced the melanoma uptake of (99m)Tc-RAD-Lys-(Arg(11))CCMSH compared to (99m)Tc-RGD-Lys-(Arg(11))CCMSH in B16/F1 melanoma-bearing C57 mice, providing a new insight into the design of α-MSH peptides for melanoma targeting.     

A Gly/Ala switch contributes to high affinity binding of benzoxazinone-based non-peptide oxytocin receptor antagonists

Non-peptide antagonists of the oxytocin receptor (OTR) have been developed to prevent pre-term labour. The benzoxazinone-based antagonists L-371,257 and L-372,662 display pronounced species-dependent pharmacology with respect to selectivity for the OTR over the V(1a) vasopressin receptor. Examination of receptor sequences from different species identified Ala(318) in helix 7 of the human OTR as a candidate discriminator required for high affinity binding. The mutant receptor [A318G]OTR was engineered and characterised using ligands representing many different chemical classes. Of all the ligands investigated, only the benzoxazinone-based antagonists had decreased affinity for [A318G]OTR. Molecular modelling revealed that Ala(318) provides a direct hydrophobic contact with a methoxy group of L-371,257 and L-372,662.     

Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction.     

The crystal structure of Pseudomonas aeruginosa lipoxygenase Ala420Gly mutant explains the improved oxygen affinity and the altered reaction specificity

Secreted LOX from Pseudomonas aeruginosa (PA-LOX) has previously been identified as arachidonic acid 15S-lipoxygenating enzyme. Here we report that the substitution of Ala420Gly in PA-LOX leads to an enzyme variant with pronounced dual specificity favoring arachidonic acid 11R-oxygenation. When compared with other LOX-isoforms the molecular oxygen affinity of wild-type PA-LOX is 1–2 orders of magnitude lower (Km O₂ of 0.4 mM) but Ala420Gly exchange improved the molecular oxygen affinity (Km O₂ of 0.2 mM). Experiments with stereo-specifically deuterated linoleic acid indicated that the formation of both 13S- and 9R-HpODE involves abstraction of the proS-hydrogen from C11 of the fatty acid backbone. To explore the structural basis for the observed functional changes (altered specificity, improved molecular oxygen affinity) we solved the crystal structure of the Ala420Gly mutant of PA-LOX at 1.8 Å resolution and compared it with the wild-type enzyme. Modeling of fatty acid alignment at the catalytic center suggested that in the wild-type enzyme dioxygen is directed to C15 of arachidonic acid by a protein tunnel, which interconnects the catalytic center with the protein surface. Ala420Gly exchange redirects intra-enzyme O₂ diffusion by bifurcating this tunnel so that C11 of arachidonic acid also becomes accessible for O₂ insertion.     

Hydrogen-exchange stabilities of RNase T1 and variants with buried and solvent-exposed Ala --> Gly mutations in the helix

Hydrogen-exchange rates were measured for RNase T1 and three variants with Ala --> Gly substitutions at a solvent-exposed (residue 21) and a buried (residue 23) position in the helix: A21G, G23A, and A21G + G23A. These results were used to measure the stabilities of the proteins. The hydrogen-exchange stabilities (DeltaG(HX)) for the most stable residues in each variant agree with the equilibrium conformational stability measured by urea denaturation (DeltaG(U)), if the effects of D(2)O and proline isomerization are included [Huyghues-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Nat. Struct. Biol. 6, 210-212]. These residues also show similar changes in DeltaG(HX) upon Ala --> Gly mutations (DeltaDeltaG(HX)) as compared to equilibrium measurements (DeltaDeltaG(U)), indicating that the most stable residues are exchanging from the globally unfolded ensemble. Alanine is stabilizing compared to glycine by 1 kcal/mol at a solvent-exposed site 21 as seen by other methods for the RNase T1 protein and peptide helix [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3833-2837], while it is destabilizing at the buried site 23 by the same amount. For the A21G variant, only local NMR chemical shift perturbations are observed compared to RNase T1. For the G23A variant, large chemical shift changes are seen throughout the sequence, although X-ray crystal structures of the variant and RNase T1 are nearly superimposable. Ala --> Gly mutations in the helix of RNase T1 at both helical positions alter the native-state hydrogen-exchange stabilities of residues throughout the sequence.     

Conformations of Gly(n)H+ and Ala(n)H+ peptides in the gas phase

High-resolution ion mobility measurements and molecular dynamics simulations have been used to probe the conformations of protonated polyglycine and polyalanine (Gly(n)H and Ala(n)H+, n = 3-20) in the gas phase. The measured collision integrals for both the polyglycine and the polyalanine peptides are consistent with a self-solvated globule conformation, where the peptide chain wraps around and solvates the charge located on the terminal amine. The conformations of the small peptides are governed entirely by self-solvation, whereas the larger ones have additional backbone hydrogen bonds. Helical conformations, which are stable for neutral Alan peptides, were not observed in the experiments. Molecular dynamics simulations for Ala(n)H+ peptides suggest that the charge destabilizes the helix, although several of the low energy conformations found in the simulations for the larger Ala(n)H+ peptides have small helical regions.     

Conformation specificity and arene binding in a peptide composed only of Lys, Ile, Ala and Gly

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aβ_1–40, a mostly disordered polypeptide implicated in Alzheimer’s disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.     

Normalization of Epigenetic Change in the Genome by Peptide Bioregulator (Ala–Glu–Asp–Gly) in Pulmonary Tuberculosis

International Journal of Peptide Research and Therapeutics - The aim of this study was to evaluate genetic and epigenetic variation of the genome in patients with sensitive pulmonary tuberculosis...     

Novel mutations (Asn 484 Lys,Thr 500 Ala,Gly 438 Glu) in Morquio B disease

Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date.We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.     

Conversion of the cleavage specificity of subtilisin YaB on oxidized insulin chains to an elastase-like specificity by replacement of Gly124 with Ala

Replacement of Gly124 on the S1 pocket of subtilisin YaB with Ala changed the cleavage pattern on oxidized insulin B-chain from the subtilisin type to the elastase type. The initial cleavage site in the B-chain shifted from L15-Y16 for wild-type YaB to A14-L15 for the G124A mutant. Upon complete hydrolysis with the G124A mutant, four of the six major cleavage sites on the B-chain were identical to porcine pancreatic elastase cleavage sites.     

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20-->Cys/Ala27-->Cys, Ser30-->Pro, Gly137-->Ala, 311-4 loop, Ser411-->Ala and Ser436-->Pro

Six previously constructed and nine newly constructed Aspergillus awamori glucoamylases with multiple mutations made by combining existing single mutations were tested for their ability to produce glucose from maltodextrins. Multiple mutations have cumulative effects on glucose yield, specific activity and thermostability. No general correlation between glucose yield and thermostability was observed, although mutations that presumably impede unfolding at high temperatures uniformly increase thermostability and generally increase glucose yield. Peak glucose yields decrease with increasing temperature. The best combination of high glucose yield, high specific activity and high thermostability occurs in Asn20-->Cys/Ala27-->Cys/Ser30-->Pro/Gly137-->Ala glucoamylase.     

Substitution of Met-69 by Ala or Gly in TEM-1 beta-lactamase confer an increased susceptibility to clavulanic acid and other inhibitors

In some inhibitor-resistant TEM-derived beta-lactamases, Met-69 is substituted by Leu, Ile or Val. Residue 69 is located in a region of strong structural constraints, at the beginning of H2 alpha-helix, and in the vicinity of B3 and B4 beta-strands. Analysis of the three-dimensional structure of TEM-1 beta-lactamase suggests that alteration of the substrate-binding site can be produced by changes of the size of residue 69 side chain. Met-69 was substituted by alanine or glycine in TEM-Bs beta-lactamase (a TEM-1-related enzyme) using site-directed mutagenesis. The minimum inhibitory concentrations of the mutants compared with the wild-type revealed an increased susceptibility to beta-lactamase inhibitor-beta-lactam combinations and to first-generation cephalosporins. Comparing the Met69Ala and Met69Gly beta-lactamases with TEM-Bs, K(m) constants of the mutants showed an increased affinity for most beta-lactams but the kcat for most substrates did not change substantially. Mutants also demonstrated lower IC50 for the three inhibitors (clavulanic acid, tazobactam and sulbactam). The two substitutions of the residue 69 by alanine and glycine had a noticeable effect on K(m) values of TEM-Bs beta-lactamase, and on affinity for beta-lactamase inhibitors.     

Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.     

A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant.

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.     

Ala analogues of the cyclolinopeptide A

Analogues of cyclolinopeptide A, due to the replacement of each amino acid in the Pro1-Pro2-Phe3-Phe4 sequences with an L-Ala residue, were synthesized by classical method in solution. Mixed anhydride and N,N'-dicyclohexylcarbodiimide coupling methods have been used for the synthesis of both linear and cyclic peptides. The products were characterized by Rf values and uv spectra, as well as by fast atom bombardment spectroscopy. 1H-nmr studies on [Ala2] analogues are also reported. Preliminary data in CDCl3 solution, at low temperature, seems more promising.     

Conformation dynamics of cyclic disulfides and selenosulfides in CXXC(U) (X = Gly,Ala) tetrapeptide redox motifs

Thioredoxin fold proteins often contain a Cys‐(Xxx)_n‐Cys(Sec) or CX_nC(U) motif, where the active cysteine (C) or selenocysteine (U) is bridged by X residues, which vary with protein function. The effect of the X residues on the conformation space of the oxidized disulfide and selenosulfide forms of the CXXC(U) motif has been investigated using molecular dynamics (MD) and density functional theory. Multi‐microsecond‐length MD simulations of the CGGC, CGAC, and CAGC cyclic peptides show that CGGC rings readily exchange between several conformations over the course of the simulation, but steric interactions with the methyl group of Ala limit the conformation space available to the cyclic peptide, especially for CGAC. The potential for the motif to be reduced, as measured by the energy of the lowest unoccupied molecular orbitals, is dependent upon the ring conformation. These results suggest that control of available conformations by the bridging residues and the protein tertiary structure may be important for defining the function of the CXXC motif. Theoretical ⁷⁷Se chemical shifts of the selenosulfide moiety are dependent upon the conformation and/or intramolecular Se···O interactions with the backbone carbonyl group of the C‐terminal U residue.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.