Valine Side Chains Research Articles

An Editing Activity That Prevents Mistranslation and Connection to Disease

I believe that the genetic code was the greatest discovery of the 20th century (Fig. 1). The code was worked out during the period that I was a student: a medical student at Tufts University (I dropped out after a year), a graduate student at MIT and Cornell (with Gordon Hammes), and a postdoctoral student at Stanford University (with Paul Flory). My research as a student was centered on theoretical and experimental physical chemistry, as applied to biological systems. With Hammes, I had done a spectroscopic and thermodynamic analysis of ribonuclease and its interaction with one of its ligands. I also had done both experimental and theoretical analyses of kinetic relaxation spectroscopy, inspired in part by the pioneering work of Eigen, who himself had been a mentor of Hammes. (Eigen gave lectures at Cornell that I attended, and we spent time in conversation as well. In more recent years, we have seen each other on a regular basis and continued our conversations.) With Flory at Stanford, I worked on statistical mechanics of polypeptides. All of that graduate and postdoctoral work was exciting, particularly the mathematical side that I especially loved because of its elegance, rigor, and abstraction. However, while this sort of mental activity was going on, I kept thinking about the newly discovered genetic code and aminoacyl-tRNA synthetases.

Vesicles from Peptidic Side-Chain Polymers Synthesized by Atom Transfer Radical Polymerization

Block copolymers can adopt a wide range of morphologies in dilute aqueous solution. There is a significant amount of interest in the use of block copolymer vesicles for a number of applications. We show that a series of oligo(valine) and oligo(phenylalanine) peptides coupled to a methacrylic group can be prepared by conventional peptide coupling techniques. These can be successfully polymerized by atom transfer radical polymerization (ATRP) in hexafluoroisopropanol (HFIP) giving access to poly(ethylene oxide)- b-poly(side-chain peptides). Many of these polymers self-assemble to form vesicles using an organic to aqueous solvent exchange. One example with a divaline hydrophobic block gives a mixture of toroids and vesicles. Circular dichroism demonstrates that secondary structuring is observed in the hydrophobic region of the vesicle walls for the valine side-chain containing polymers.

Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase

NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

Nucleation of an Allosteric Response via Ligand-induced Loop Folding

The Escherichia coli biotin repressor BirA is an allosteric transcription regulatory protein to which binding of the small ligand corepressor biotinyl-5′-AMP promotes homodimerization and subsequent DNA binding. Structural data indicate that the apo or unliganded repressor is characterized by four partially disordered loops that are ordered in the ligand-bound dimer. While three of these loops participate directly in the dimerization, the fourth, consisting of residues 212–234 is distal to the interface. This loop, which is ordered around the adenine ring of the adenylate moiety in the BirA adenylate structure, is referred to as the adenylate-binding loop (ABL). Although residues in the loop do not interact directly with the ligand, a hydrophobic cluster consisting of a tryptophan and two valine side-chains assembles over the adenine base. Results of previous measurements suggest that folding of the ABL is integral to the allosteric response. This idea and the role of the hydrophobic cluster in the process were investigated by systematic replacement of each side-chain in the cluster with alanine and analysis of the mutant proteins for small ligand binding and dimerization. Isothermal titration calorimetry measurements indicate defects in adenylate binding for all ABL variants. Additionally, sedimentation equilibrium measurements reveal that coupling between adenylate binding and dimerization is compromised in each mutant. Partial proteolysis measurements indicate that the mutants are defective in ligand-linked folding of the ABL. These results indicate that the hydrophobic cluster is critical to the ligand-induced disorder-to-order transition in the ABL and that this transition is integral to the allosteric response in the biotin repressor.

A Parallel Coiled-Coil Tetramer with Offset Helices,

Specific helix-helix interactions are fundamental in assembling the native state of proteins and in protein-protein interfaces. Coiled coils afford a unique model system for elucidating principles of molecular recognition between alpha helices. The coiled-coil fold is specified by a characteristic seven amino acid repeat containing hydrophobic residues at the first (a) and fourth (d) positions. Nonpolar side chains spaced three and four residues apart are referred to as the 3-4 hydrophobic repeat. The presence of apolar amino acids at the e or g positions (corresponding to a 3-3-1 hydrophobic repeat) can provide new possibilities for close-packing of alpha-helices that includes examples such as the lac repressor tetramerization domain. Here we demonstrate that an unprecedented coiled-coil interface results from replacement of three charged residues at the e positions in the dimeric GCN4 leucine zipper by nonpolar valine side chains. Equilibrium circular dichroism and analytical ultracentrifugation studies indicate that the valine-containing mutant forms a discrete alpha-helical tetramer with a significantly higher stability than the parent leucine-zipper molecule. The 1.35 A resolution crystal structure of the tetramer reveals a parallel four-stranded coiled coil with a three-residue interhelical offset. The local packing geometry of the three hydrophobic positions in the tetramer conformation is completely different from that seen in classical tetrameric structures yet bears resemblance to that in three-stranded coiled coils. These studies demonstrate that distinct van der Waals interactions beyond the a and d side chains can generate a diverse set of helix-helix interfaces and three-dimensional supercoil structures.

Structural Basis for Substrate Recognition by the Editing Domain of Isoleucyl-tRNA Synthetase

In isoleucyl-tRNA synthetase (IleRS), the "editing" domain contributes to accurate aminoacylation by hydrolyzing the mis-synthesized intermediate, valyl-adenylate, in the "pre-transfer" editing mode and the incorrect final product, valyl-tRNA(Ile), in the "post-transfer" editing mode. In the present study, we determined the crystal structures of the Thermus thermophilus IleRS editing domain complexed with the substrate analogues in the pre and post-transfer modes, both at 1.7 A resolution. The active site accommodates the two analogues differently, with the valine side-chain rotated by about 120 degrees and the adenosine moiety oriented upside down. The substrate-binding pocket adjusts to the adenosine-monophosphate and adenosine moieties in the pre and post-transfer modes, respectively, by flipping the Trp227 side-chain by about 180 degrees . The substrate recognition mechanisms of IleRS are characterized by the active-site rearrangement between the two editing modes, and therefore differ from those of the homologous valyl and leucyl-tRNA synthetases from T.thermophilus, in which the post-transfer mode is predominant. Both modes of editing activities were reduced by replacements of Trp227 with Ala, Val, Leu, and His, but not by those with Phe and Tyr, indicating that the aromatic ring of Trp227 is important for the substrate recognition. In both editing modes, Thr233 and His319 recognize the substrate valine side-chain, regardless of the valine side-chain rotation, and reject the isoleucine side-chain. The T233A and H319A mutants have detectable editing activities against the cognate isoleucine.

Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hydrophobic Heptad Repeat

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.

The role of backbone stability near Ala44 in the high reduction potential class of rubredoxins

Rubredoxins may be separated into high and low reduction potential classes, with reduction potentials differing by approximately 50 mV. Our previous work showed that a local shift in the polar backbone due to an A(44) versus V(44) side-chain size causes this reduction potential difference. However, this work also indicated that in the low potential Clostridium pasteurianum (Cp) rubredoxin, a V(44) --> A(44) mutation causes larger local backbone flexibility, because the V(44) side-chain present in the wild-type (wt) is no longer present to interlock with neighboring residues to stabilize the subsequent G(45). Since Pyrococcus furiosus (Pf) and other high potential rubredoxins generally have a P(45), it was presumed that a G(45) --> P(45) mutation might stabilize a V(44) --> A(44) mutation in Cp rubredoxin. Here crystal structure analysis, energy minimization, and molecular dynamics (MD) were performed for wt V(44)G(45), single mutant A(44)G(45) and double mutant A(44)P(45) Cp, and for wt A(44)P(45) Pf rubredoxins. The local structural, dynamical, and electrostatic properties of Cp gradually approach wt Pf in the order wt Cp to single to double mutant because of greater sequence similarity, as expected. The double mutant A(44)P(45) Cp exhibits increased backbone stability near residue 44 and thus enhances the probability that the backbone dipoles point toward the redox site, which favors an increase in the electrostatic contribution to the reduction potential. It appears that the electrostatic potential of residue 44 and the solvent accessibility to the redox are both determinants for the reduction potentials of homologous rubredoxins. Overall, these results indicate that an A(44) in a rubredoxin may require a P(45) for backbone stability whereas a V(44) can accommodate a G(45), since the valine side-chain can interlock with its neighbors.

Studies of helix fraying and solvation using 13C′ isotopomers

Both NMR and IR studies of carbonyl (13C') isotopomers of designed helices can provide residue-level details regarding the fractional occurrence and melting behavior of helical phi/psi angles along the sequence of helical peptides, details that cannot be obtained from CD or 1H-NMR studies. We have studied a classic series of helical models, Ac-YGG-(KAXAA)3K-NH2 (X=A,V), in both aqueous and helix-favoring media containing fluoroalcohol cosolvents, including a solvent system allowing the observation of cold denaturation. These studies confirmed the strong N-capping associated with this sequence and revealed more extensive C-terminal fraying than that calculated using current helicity prediction algorithms. In the X=A series, the central residues are somewhat resistant to thermal melting; it instead occurs predominantly at the frayable C terminus. For the X=V series under cold-denaturing conditions, the temperature of maximal helicity is not uniform along the sequence and both solvated and nonsolvated helical alanine sites (13C=O stretches at 1592 cm(-1) and 1615 cm(-1), respectively) are apparent. Correlation between the two spectroscopies employed yielded the intriguing observation that the valine side chain is able to desolvate the i - 4 amide in short monomeric helices. In addition, we report further measurements of the temperature dependence of alanine statistical coil chemical shifts, the temperature dependence of the 13C chemical shift of urea (employed as chemical shift reference), and a useful formula for converting 13C' shifts into fractional helicities.

Novel Interaction Partners of the CD2BP2-GYF Domain

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B' were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis.

A Role for the 2′ Residue in the Second Transmembrane Helix of the GABAA Receptor γ2S Subunit in Channel Conductance and Gating

GABA(A) receptors composed of alpha, beta and gamma subunits display a significantly higher single-channel conductance than receptors comprised of only alpha and beta subunits. The pore of GABA(A) receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6', 10' and 13' positions. At the 2' position, however, a polar residue is present in the gamma subunit but not the alpha or beta subunits. As residues at the 2', 6' and 10' positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type alpha1beta1 and alpha1beta1gamma2S receptors with two mutant receptors, alphabetagamma(S2'A) and alphabetagamma(S2'V). We found that the single-channel conductance of both mutant alphabetagamma receptors was significantly decreased with respect to wild-type alphabetagamma, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant alphabetagamma receptors remained larger than wild-type alphabeta channels. This reduction in the conductance of mutant alphabetagamma receptors was observed at depolarized potentials only (E(Cl) = -1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the gamma2' residue. The substitutions at the gamma2' serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2' position in M2 of the gamma subunit affects both single-channel conductance and receptor kinetics.

Crystal Structure and Conformation of N-(t-Butoxycarbonyl)-L-Isoleucyl-L-Valine Methyl Ester (Boc-Ile-Val-OMe)†

ABSTRACT N-(t-Butoxycarbonyl)-L-Isoleucyl-L-Valine Methyl ester, C17H32N2O5, F.W.=344.46, crystallizes in hexagonal space group P65 with parameters, a=b = 20.285(5), c=27.160(5) A˚, V=9679.1(6) A˚ 3, Z = 18, Dcal=1.064 Mg cm−3, λ(CuKα) = 1.5418 A˚, μ (CuKα). The dipeptide Boc-Ile-Val-OMe has three crystallographically independent molecules (A, B, and C) in the asymmetric unit. The peptide units are in trans conformation and the three molecules adopt an extended conformation. The side chains of isoleucine and valine for the three molecules A, B, and C take up g− t, g− g−, and g− t; and g− g+, g+ t, and tg− conformations, respectively. The molecules form an infinite ribbon of β-sheet structure, which in turn forms helical arrangement along the c-direction. They are stabilized by N − H … O and C − H … O types of intermolecular interactions.

L-Valyl-L-serine trihydrate

The valine side chains in the crystal structure of the title compound [systematic name: 2-(2-ammonio-3-methylbutanamido)-3-hydroxypropanoate trihydrate], C8H16N2O4.3H2O, stack along an a axis of 4.77 A to form hydrophobic columns surrounded by remarkable water/hydroxyl shells. The peptide main chains are connected by hydrogen bonds in two-dimensional layers. The peptide molecules in each layer are related only by translation, and generate a very rare pattern. This is rendered possible through the formation of the shortest C(alpha)-H...O(carboxylate) interaction ever recorded.

L-Asparaginyl-L-valine 1.33-hydrate

The title compound, C9H17N3O4·1.33H2O, crystallizes with three peptide mol­ecules and four water mol­ecules in the asymmetric unit. Two of the peptide mol­ecules contain an intramolecular hydrogen bond between the N-terminal amino group and the asparagine side chain. Valine side chains aggregate into large hydro­phobic columns.

Role of a Surface Tryptophan in Defining the Structure, Stability, and DNA Binding of the Hyperthermophile Protein Sac7d,

Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein-DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (DeltaG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 degrees C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.

Do Valine Side Chains Have an Influence on the Folding Behavior of β‐Substituted β‐Peptides?

AbstractThe influence of valine side chains on the folding/unfolding equilibrium and, in particular, on the 314‐helical propensity of β3‐peptides were investigated by means of molecular‐dynamics (MD) simulation. To that end, the valine side chains in two different β3‐peptides were substituted by leucine side chains. The resulting four peptides, of which three have never been synthesized, were simulated for 150 to 200 ns at 298 and 340 K, starting from a fully extended conformation. The simulation trajectories obtained were compared with respect to structural preferences and folding behavior. All four peptides showed a similar folding behavior and were found to predominantly adopt 314‐helical conformations, irrespective of the presence of valine side chains. No other well‐defined conformation was observed at significant population in any of the simulations. Our results imply that β3‐peptides show a structural preference for 314‐helices independent of the branching nature of the side chains, in contrast to what has been previously proposed on the basis of circular‐dichroism (CD) measurements.

Crystal Structures of the CP1 Domain from Thermus thermophilus Isoleucyl-tRNA Synthetase and Its Complex with l-Valine

Isoleucyl-tRNA synthetase (IleRS) links tRNA(Ile) with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNA(Ile). We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-A resolutions, respectively. In the complex structure, the Asp(328) residue, which was shown to be critical for the editing reaction against Val-tRNA(Ile) by a previous mutational analysis, recognizes the valine NH(3)(+) group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His(319). The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNA(Ile) in addition to Val-tRNA(Ile), indicating that the valine-binding mode found in this study represents that in the Val-tRNA(Ile) editing reaction. Analyses of the Val-tRNA(Ile) editing activities of T. thermophilus IleRS mutants revealed the importance of Thr(228), Thr(229), Thr(230), and Asp(328), which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNA(Ile) bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized.

2′‐Fluoro‐4′‐thio‐2′,3′‐unsaturated Nucleosides: anti‐HIV Activity, Resistance Profile, and Molecular Modeling Studies

AbstractFor Abstract see ChemInform Abstract in Full Text.

2′-Fluoro-4′-thio-2′,3′-unsaturated Nucleosides: Anti-HIV Activity, Resistance Profile, and Molecular Modeling Studies

Both D- and L-2′-fluoro-4′-thio-2′,3′-unsaturated nucleosides were synthesized and their anti-HIV activity against the drug sensitive virus and lamivudine-resistant mutant (M184V) were evaluated. In vitro antiviral evaluation indicated that the L-isomers are more potent than the D-isomers, but unfortunately all were cross-resistant with 3TC. Molecular modeling studies revealed that the unnatural sugar moiety of the L-nucleosides as well as 4′-sulfur atom of the D-isomer has a steric conflict with the bulky side chain of valine 184, resulting in cross-resistance.

Conformational effects of the valine sidechain on the β Lβ L extended and Type I beta turn backbone structures of MeCO-Val-Ala-NHMe and MeCO-Ala-Val-NHMe. An ab initio exploratory conformational study

Ab initio molecular studies were carried out on two dipeptide models, MeCO-Ala-Val-NHMe and MeCO-Val-Ala-NHMe at the RHF/3-21G level of theory. All possible sidechain conformations were investigated for the extended β Lβ L and Type I beta turn (α Lδ L) backbone structures. The relative stabilities of the sidechain conformers and also the topologies of the PEC-s obtained by rotating the valine sidechain are discussed. Our exploratory study shows that these curves of the two isomeric dipeptides are almost identical in the case of the β Lβ L extended backbone conformations, while in the case of the α Lδ L backbone conformations they are completely different. The results also show a considerable effect of the rotation of the valine sidechain on the backbone dihedral angles, the magnitude of this effect varies in the different backbone conformations.