Size Of Side Chain Research Articles

The HAMP Domain Structure Implies Helix Rotation in Transmembrane Signaling

HAMP domains connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases. The solution structure of an archaeal HAMP domain shows a homodimeric, four-helical, parallel coiled coil with unusual interhelical packing, related to the canonical packing by rotation of the helices. This suggests a model for the mechanism of signal transduction, in which HAMP alternates between the observed conformation and a canonical coiled coil. We explored this mechanism in vitro and in vivo using HAMP domain fusions with a mycobacterial adenylyl cyclase and an E. coli chemotaxis receptor. Structural and functional studies show that the equilibrium between the two forms is dependent on the side-chain size of residue 291, which is alanine in the wild-type protein.

Role of main chain rigidity and side-chain substitution on the supramolecular organization of rigid–flexible polymers

The synthesis, characterization and supramolecular organization of rigid–flexible aromatic–aliphatic polyethers are reported. The polymers contain substituted quinquephenyl moieties along their main chains separated by flexible aliphatic units of various lengths. The oligophenylene segments bear different types of dodecyloxy tailed side groups, either attached on the periphery of side dendritic wedges (G 1) or as direct side-chains (G 0). Their structural characterization was performed by means of differential scanning calorimetry, polarizing optical microscopy and X-ray scattering from oriented fibers. These techniques revealed the presence of supramolecular organization in different levels and in the absence of solvent, reflecting a delicate balance of interactions produced by the rigid–flexible backbone segments and the size of dendritic side-chains. It was concluded that the polymers with only two dodecyloxy side-chains (zeroth generation, G 0) possess smectic order. On the other hand, substitution of the quinquephenyl backbone with the dodecyloxy decorated dendrons (first generation, G 1) disrupts the lateral order giving rise to a nematic state. These results are compared with the corresponding rigid–flexible polyethers having terphenyl rigid segments.

Engineering of a Single Conserved Amino Acid Residue of Herpes Simplex Virus Type 1 Thymidine Kinase Allows a Predominant Shift from Pyrimidine to Purine Nucleoside Phosphorylation

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).

Synthesis of Piperazinephen-1-ylethylamines as Potent and Selective Antagonists of the Human Melanocortin-4 Receptor

A series of piperazinephen-1-ylehtylamines were synthesized and evaluated for their biological activity at the human melanocortin-4 receptor. This modification reduced the size and flexibility of the N-alkyl side-chain of some earlier lead compounds, while maintained the low nanomolar binding affinity. Keywords: Melanocortin, antagonist, piperazinephenylamine, synthesis

Modelling the interaction of several bisphosphonates with hydroxyapatite using the generalised AMBER force field

The ability of the Generalised AMBER Force Field (GAFF) of Kollman and co-workers to model the structures of bisphosphonate ligands, C(R 1)(R 2)(PO 3 2−) 2, important compounds in the treatment of bone cancer, by molecular mechanics methods is evaluated. The structure of 50 bisphosphonates and nine bisphosphonate esters were predicted and compared to their crystal structures. Partial charges were assigned from a RHF/6-31G ∗ single point calculation at the geometry of the crystal structure. Additional parameters required for GAFF were determined using the methods of the force field’s developers. The structures were found to be well replicated with virtually all bond lengths reproduced to within 0.015 Å, or within 1.2 σ of the crystallographic mean. Bond angles were reproduced to within 1.9° (0.8 σ). The observed gauche or anti conformation of the molecules was reproduced, although in several instances gauche conformations observed in the solid state energy-minimised into anti conformations, and vice versa. The interaction of MDP (R 1 = R 2 = H), HEDP (R 1 = OH, R 2 = CH 3), APD (R 1 = OH, R 2 = (CH 2) 2NH 3 +), alendronate (R 1 = OH, R 2 = (CH 2) 3NH 3 +) and neridronate (R 1 = OH, R 2 = (CH 2) 5NH 3 +) with the (001), (010) and (100) faces of hydroxyapaptite was examined by energy-minimising 20 random orientations of each ligand 20 Å from the mineral (where there is no interaction), and then at about 8 Å from the surface whereupon the ligand relaxes onto the surface. The difference in energy between the two systems is the interaction energy. In all cases interaction with hydroxyapatite caused a decrease in energy. When modelled with a dielectric constant of 78 ε o, non-bonded interactions dominate; electrostatic interactions become important when the dielectric constant is <10 ε o. Irrespective of the value of the dielectric constant used, the structure of the ligands on the hydroxyapatite surface is very similar. On the (001) face, both phosphonate groups interact near a surface Ca 2+ ion. The magnitude of the exothermic interaction energy varies with molecular volume (MDP<HEDP<APD<alendronate) except for neridronate which interacts less effectively than alendronate because the long amino side chain folds in on itself and does not align with the surface of the mineral. The bisphosphonates adopt two conformations on the (010) face. In the first of these, found for MDP and 40% of the alendronate structures, both phosphonates interact with the surface and the side chain points away from the surface. Hence, the interaction energy is similar for both species. In the second conformation, adopted by the majority of ligands, one phosphonate and the Cα side chain interact with the surface. The interaction energy, the magnitude of which is very similar to that on the (001) face, increases with the molecular volume of the ligand, again with the exception of neridronate. Two conformations also occur on the (100) face. In the first conformation, only one of the phosphonate groups points towards the surface and the Cα side chain interacts with the surface; in the second conformation the Cα side chain interacts strongly with the surface and both phosphonate groups point away from the surface towards the solution. The first conformation, which is the more common, is energetically more favourable. Its magnitude is virtually insensitive to the nature of the side chain and is similar to the magnitude of the interaction energy on the other two faces. The magnitude of the second conformation increases with the size of the Cα side chain.

Microcalorimetry of interaction of dihydro-imidazo-phenanthridinium (DIP)-based compounds with duplex DNA

Isothermal titration (ITC) and differential scanning calorimetry (DSC) have been used to screen the binding thermodynamics of a family of DNA intercalators based on the dihydro-imidazo-phenanthridinium (DIP) framework. All members of this DIP-based ligand family bind to both genomic (calf thymus and/or salmon testes) and a synthetic dodecamer d(CGCGAATTCGCG) duplex DNA with broadly similar affinities regardless of side chain size or functionality. Viscosity measurements confirm that binding satisfies standard criteria for intercalation. Binding is exothermic but with an additional favourable positive entropy contribution in most cases at 25 °C, although a significant negative heat capacity effect (Δ C p) means that both Δ H° and Δ S° decrease with increasing temperature. DIP-ligand binding to DNA also shows significant entropy–enthalpy compensation effects that are now almost standard in such situations, probably reflecting the conformational flexibility of macromolecular systems involving a multiplicity of weak non-covalent interactions. This ability to vary side chain functionality without compromising DNA binding suggests that the DIP framework should be a promising basis for more adventurous chemistry at the DNA level.

Synthesis and Characterization of Poly(fluorene)-Based Copolymers Containing Various 1,3,4-Oxadiazole Dendritic Pendants

A series of poly(fluorene)s (PFs) containing different generations of poly(benzyl ether) dendritic wedges with oxadiazole (OXD) peripheral functional groups, including copolymers bearing carbazole (CAZ) pendent groups, were synthesized. The dendritic side-chain polymers possessed excellent solubility in common solvents and good thermal stability with decomposition temperatures of 5% weight loss at more than 370 °C. Photophysical studies reveal that the photoluminescent (PL) properties of the dendronized polymers were greatly affected by the size of the dendritic side chains. The G1- and G2-substituted PF derivatives greatly suppress the aggregation PF backbones and thus to induce pure blue PL emission (with shorter wavelengths than PF). Excitation of the peripheral functional groups leads to substantial energy transfer from the dendrons to the poly(fluorene) backbones. In addition, the excitation of PF bearing G2 dendrons (G2-OXD) at 303 nm creates stronger fluorescence than that at 390 nm, indicating tha...

The Role of Protein Dynamics in Thymidylate Synthase Catalysis: Variants of Conserved 2‘-Deoxyuridine 5‘-Monophosphate (dUMP)-Binding Tyr-261,

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

Peptide-Binding Motifs for the I-Ad MHC Class II Molecule: Alternate pH-Dependent Binding Behavior

The ability of peptides to form stable complexes with MHC class II molecules expressed in the host determines their ability to recruit CD4 T cells during an immune response. In this study, we sought to define the features of the antigenic peptides that control their kinetic stability with I-A(d) because of the diversity of peptides that this molecule is known to present. Peptide dissociation assays indicated that each pocket of I-A(d) displays exquisite sensitivity to side chain structure, size, and charge. Most surprising were results related to the P1 pocket, which has been difficult to define by conventional competition assays. Our studies revealed a considerable degree of specificity in the P1 pocket but also an unexpected degree of structural flexibility. Amino acids with neutral side chains such as Met and the alternatively negatively charged Glu are both highly favored at P1. Interestingly, these two options at the P1 pocket in I-A(d) display dramatically different pH-dependent interactions with the class II molecule. These findings are discussed in the context of a structural model to explain these data and in light of the immunological implications of pH-dependent behavior of class II-peptide complexes in acidic endosomal compartments, where DM-catalyzed loading of class II molecules takes place, and at the neutral pH of the APC cell surface, where class II-peptide complexes promote activation of CD4 T cells.

Structural Definition of the H-2Kd Peptide-binding Motif

Classic major histocompatibility complex (MHC) proteins associate with antigen- and self-derived peptides in an allele-specific manner. Herein we present the crystal structure of the MHC class I protein H-2K(d) (K(d)) expressed by BALB/c mice in complex with an antigenic peptide derived from influenza A/PR/8/34 nucleoprotein (Flu, residues 147-155, TYQRTRALV). Analysis of our structure in conjunction with the sequences of naturally processed epitopes provides a comprehensive understanding of the dominant K(d) peptide-binding motif. We find that Flu residues Tyr(P2), Thr(P5), and Val(P9) are sequestered into the B, C, and F pockets of the K(d) groove, respectively. The shape and chemistry of the polymorphic B pocket make it an optimal binding site for the side chain of Tyr(P2) as the dominant anchoring residue of nonameric peptides. The non-polar F pocket limits the amino acid repertoire at P9 to hydrophobic residues such as Ile, Leu, or Val, whereas the C pocket restricts the size of the P5-anchoring side chain. We also show that Flu is accommodated in the complex through an unfavorable kink in the otherwise extended peptide backbone due to the presence of a prominent ridge in the K(d) groove. Surprisingly, this backbone conformation is strikingly similar to D(b)-presented peptides despite the fact that these proteins employ distinct motif-anchoring strategies. The results presented in this study provide a solid foundation for the understanding of K(d)-restricted antigen presentation and recognition events.

Grow to Fit Molecular Dynamics (G2FMD): an ab initio method for protein side-chain assignment and refinement

The rough energy landscapes and tight packing of protein interiors are two of the critical factors that have prevented the wide application of physics-based models in protein side-chain assignment and protein structure prediction in general. Complementing the rotamer-based methods, we propose an ab initio method that utilizes molecular mechanics simulations for protein side-chain assignment and refinement. By reducing the side-chain size, a smooth energy landscape was obtained owing to the increased distances between the side chains. The side chains then gradually grow back during molecular dynamics simulations while adjusting to their surrounding driven by the interaction energies. The method overcomes the barriers due to tight packing that limit conformational sampling of physics-based models. A key feature of this approach is that the resulting structures are free from steric collisions and allow the application of all-atom models in the subsequent refinement. Tests on a small set of proteins showed nearly 100% accuracy on both chi1 and chi2 of buried residues and 94% of them were within 20 degrees from the native conformation, 79% were within 10 degrees and 42% were within 5 degrees . However, the accuracy decreased when exposed side chains were involved. Further improvement and application of the method and the possible reasons that affect the accuracy on the exposed side chains are discussed.

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.

Optimization of aromatic side chain size complementarity in the hydrophobic core of a designed coiled‐coil

The coiled-coil structure plays an important roles, especially in protein assembly. Previously we constructed AAB-type heterotrimeric coiled-coils by manipulating the packing in the hydrophobic core using Trp and Ala residues, where one Trp and two Ala residues were placed in the hydrophobic core instead of three Ile residues. To optimize the packing complementarity in the hydrophobic core, we investigated the effects of introducing various aromatic amino acids on the formation of an AAB-type heterotrimeric coiled-coil, by circular dichroism, thermal stability, and nuclear magnetic resonance (NMR) studies. We found that the Phe residue was more suitable for heterotrimeric coiled-coil formation than the Trp residue, when combined with two Ala residues, whereas the Tyr and His residues did not induce the coiled-coil structure efficiently.

The Position 68(E11) Side Chain in Myoglobin Regulates Ligand Capture, Bond Formation with Heme Iron, and Internal Movement into the Xenon Cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.

An Active Enzyme Constructed from a 9-Amino Acid Alphabet

Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.

Influence of isoleucyl dipeptide side-chains on the dissociation and intermolecular interactions in water at 298.15 K

Enthalpies of dissociation of four isoleucyl dipeptides having alkyl side-chains ( l-isoleucine-glycine, l-Ile-Gly; l-isoleucine- l-alanine, l-Ile- l-Ala; l-isoleucine- l-valine, l-Ile- l-Val; l-isoleucine- l-isoleucine, l-Ile- l-Ile) in water at 298.15 K have been determined. Enthalpies of dilution have also been calculated. These results were used to derive the enthalpic coefficients of interactions according to the modified McMillan–Mayer's model. The results are considered in an effort to establish a possible relation between dissociation, intermolecular interactions and the size of the alkyl side-chains.

Grouping of amino acid types and extraction of amino acid properties from multiple sequence alignments using variance maximization

Understanding of amino acid type co-occurrence in trusted multiple sequence alignments is a prerequisite for improved sequence alignment and remote homology detection algorithms. Two objective approaches were used to investigate co-occurrence, both based on variance maximization of the weighted residue frequencies in columns taken from a large alignment database. The first approach discretely grouped amino acid types, and the second approach extracted orthogonal properties of amino acids using principal components analysis. The grouping results corresponded to amino acid physical properties such as side chain hydrophobicity, size, or backbone flexibility, and an optimal arrangement of approximately eight groups was observed. However, interpretation of the orthogonal properties was more complex. Although the principal components accounting for the largest variances exhibited modest correlations with hydrophobicity and conservation of glycine, in general principal components did not correspond to physical properties of amino acids. Although not intuitive, these amino acid mathematical properties were demonstrated to be robust and to improve local pairwise alignment accuracy, relative to 20 amino acid frequencies alone, for a simple test case.

Thermoelectric Properties of Poly(3-Alkylthiophenes)

Typical conductive polymers of poly(3-alkylthiophenes) were synthesized by oxidative polymerization. Alkyl side chains were CnH2n+1 with n=4, 6, 8, 12. The regioregularity with the HT linkage was larger than 99% based on NMR analysis. Completely regioregular head-to-tail (HT) poly(3-alkylthiophenes) were obtained. We have evaluated the effect of side chain size on the thermoelectric properties of Seebeck coefficient and electrical conductivity. The results were as follows: 1) Seebeck coefficient decreased with an increasing electrical conductivity. 2) High Seebeck coefficient &gt;1mV/K was observed at low electrical conductivity &lt;10-2S/cm. 3) The small side chain caused the higher electrical conductivity in the range of electrical conductivity &lt;10- 1S/cm.

Proline mutations induce negative‐dosage effects on uptake velocity of the dopamine transporter

Ala and Gly substitutions for Pro 101 (P101) located in transmembrane domain 2 of the dopamine transporter (DAT) abolished transport activity but did not disrupt plasma membrane expression. Due to the high conservation of P101 in all neurotransmitter transporters and the capability of Pro to add flexibility to helices, we hypothesized that P101 contributes to the dynamic feature of substrate translocation. To test this hypothesis, here we analysed transport activity for DAT mutants where this Pro was mutated into different amino acids, including Ser, Val, Leu and Phe. The transmembrane domain 2 helix of P101F, unlike the other mutants, was computationally predicted to have a Van der Waals energy threefold higher than the wild-type helix. P101F mutant expression was consistently disrupted in COS cells. Among all the other mutants that express normally, P101V, with a side-chain size close to that of Pro, restores the transport activity of P101A by sevenfold. Most importantly, P101V, P101L and P101S display negative-dosage effects on dopamine (DA) transport, i.e. the velocity-concentration curve for DA uptake does not show a plateau with increasing [DA] but rather peaks and then goes down. These data support the view that P101 of DAT plays an essential role in DA translocation.

Influence of steric hindrance on enantioseparation of Dns‐amino acids and pesticides on terguride based chiral selectors in capillary electrophoresis

Three urea derivatives of ergoline-based chiral selectors (CSs), differing in the size of the urea side chain, i.e. dimethyl- (CSI), diethyl- (CSII), and diisopropylurea (CSIII), were used to study the effect of steric hindrance on the enantioseparation of dansyl amino acids (Dns-AAs), pesticides, and mandelic acid under condition of capillary electrophoresis (CE) in linear polyacrylamide coated capillaries. A mixture of organic modifiers (MeOH/THF, 4:1 v/v) in a BGE consisting of 100 mM beta-alanine-acetate was used to increase the solubility of CSs up to 25 mM. The capillary was filled with CS (high UV absorption), and the inlet and outlet vials contained buffer solutions only. The best enantioseparation of Dns-AAs was achieved on CSI. Increased steric hindrance of the chiral binding site led to reduction of both enantioselectivity and resolution. The opposite pattern was observed for the separation of mandelic acid enantiomers, where the best enantioseparation and resolution was obtained with CSIII. Most of the pesticides studied reached maximum selectivity on the diethylurea ergoline derivative (CSII). Enantioseparation of fenoxaprop was found to be independent of steric hindrance.