Mammalian neuronal sodium channel Blocker mu-conotoxin BuIIIB has a structured N-terminus that influences potency

Among the mu-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, mu-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23, and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in mu-BuIIIB, which is also present in mu-BuIIIA and mu-BuIIIC but absent in other mu-conotoxins, forms part of a short a-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related mu-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of mu-BuIIIB for Na(v)1.3. One analogue, D-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. On the basis of previous results for related mu-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated and suggest that further gains in potency might be achieved by additional modifications of this region.

An automated approach to network features of protein structure ensembles

Network theory applied to protein structures provides insights into numerous problems of biological relevance. The explosion in structural data available from PDB and simulations establishes a need to introduce a standalone-efficient program that assembles network concepts/parameters under one hood in an automated manner. Herein, we discuss the development/application of an exhaustive, user-friendly, standalone program package named PSN-Ensemble, which can handle structural ensembles generated through molecular dynamics (MD) simulation/NMR studies or from multiple X-ray structures. The novelty in network construction lies in the explicit consideration of side-chain interactions among amino acids. The program evaluates network parameters dealing with topological organization and long-range allosteric communication. The introduction of a flexible weighing scheme in terms of residue pairwise cross-correlation/interaction energy in PSN-Ensemble brings in dynamical/chemical knowledge into the network representation. Also, the results are mapped on a graphical display of the structure, allowing an easy access of network analysis to a general biological community. The potential of PSN-Ensemble toward examining structural ensemble is exemplified using MD trajectories of an ubiquitin-conjugating enzyme (UbcH5b). Furthermore, insights derived from network parameters evaluated using PSN-Ensemble for single-static structures of active/inactive states of 2-adrenergic receptor and the ternary tRNA complexes of tyrosyl tRNA synthetases (from organisms across kingdoms) are discussed. PSN-Ensemble is freely available from http://vishgraph.mbu.iisc.ernet.in/PSN-Ensemble/psn_index.html.

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis

Background: DNA-binding protein from starved cells (Dps) are nano-compartments that can oxidize and store iron rendering protection from free radicals. Results: A histidine-aspartate ionic cluster in mycobaterial Dps2 modulates the rate of iron entry and exit in these proteins. Conclusion: Substitutions that disrupt the cluster interface alter the iron uptake/release properties with localized structural changes. Significance: Identifying important gating residues can help in designing nano-delivery vehicles. Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 for the various mutants to compare structural alterations vis a vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

Allosteric Transition Induced by Mg2+ Ion in a Transactivator Monitored by SERS

We demonstrate the utility of the surface-enhanced Raman spectroscopy (SERS) to monitor conformational transitions in protein upon ligand binding. The changes in protein's secondary and tertiary structures were monitored using amide and aliphatic/aromatic side chain vibrations. Changes in these bands are suggestive of the stabilization of the secondary and tertiary structure of transcription activator protein C in the presence of Mg2+ ion, whereas the spectral fingerprint remained unaltered in the case of a mutant protein, defective in Mg2+ binding. The importance of the acidic residues in Mg2+ binding, which triggers an overall allosteric transition in the protein, is visualized in the molecular model. The present study thus opens up avenues toward the application of SERS as a potential tool for gaining structural insights into the changes occurring during conformational transitions in proteins.

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (phi, ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (phi, ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (phi, ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.

INDUCTION OF FOLDED STRUCTURES IN DESIGNED PEPTIDES USING CONFORMATIONALLY CONSTRAINED RESIDUES

Folding into compact globular structures, with well-defined modules of secondary structure, appears to be a characteristic of long polypeptide chains, with a specific patterning of coded amino acid residues along the length of sequence. Cooperative hydrogen bond driven secondary structure formation and solvent forces, which contribute favorably to the entropy of folding, by promoting compaction of the polymeric chain, have long been discussed as major determinants of the folding process. First principles design approaches, which use non-coded amino acids, employ an alternative structure directing strategy, by using amino acid residues which exhibit a strong conformational bias for specific regions of the Ramachandran map. This overview of ongoing studies in the authors' laboratory, attempts to explore the use of conformationally restricted amino acid residues in the design of peptides with well-defined secondary structures. Short peptides composed of 20 genetically coded amino acids usually exist in solution as an ensemble of equilibrating conformations. Apolar peptide sequences, which are readily soluble in organic solvents like chloroform and methanol, facilitate formation of structures which are predominately driven by intramolecular hydrogen bond formation. The choice of sequences containing residues with a limited range of conformational choices strongly favors formation of local turn structures, stabilized by short range intramolecular hydrogen bonds. Two residue beta-turns can nucleate either helical or hairpin folding, depending on the precise conformation of the turn segment Restriction of the conformational space available to amino acid residues is easily achieved by introduction of an additional alkyl group at the C alpha carbon atom or by side chain backbone cyclization, as in proline. Studies of synthetic sequences incorporating two prototype residues alpha-aminoisobutyric acid (Aib) and D-proline (DPro) illustrate the utility of the strategy in construction of helices and hairpins. Extensions to the design of conformationally switchable sequences and structurally defined hybrid peptides containing backbone homologated residues are also surveyed.

Sparsely populated residue conformations in protein structures: Revisiting ``experimental'' Ramachandran maps

The Ramachandran map clearly delineates the regions of accessible conformational (phi-) space for amino acid residues in proteins. Experimental distributions of phi, values in high-resolution protein structures, reveal sparsely populated zones within fully allowed regions and distinct clusters in apparently disallowed regions. Conformational space has been divided into 14 distinct bins. Residues adopting these relatively rare conformations are presented and amino acid propensities for these regions are estimated. Inspection of specific examples in a completely arid, fully allowed region in the top left quadrant establishes that side-chain and backbone interactions may provide the energetic compensation necessary for populating this region of phi- space. Asn, Asp, and His residues showed the highest propensities in this region. The two distinct clusters in the bottom right quadrant which are formally disallowed on strict steric considerations correspond to the gamma turn (C7 axial) conformation (Bin 12) and the i + 1 position of Type II turns (Bin 13). Of the 516 non-Gly residues in Bin 13, 384 occupied the i + 1 position of Type II turns. Further examination of these turn segments revealed a high propensity to occur at the N-terminus of helices and as a tight turn in hairpins. The strand-helix motif with the Type II turn as a connecting element was also found in as many as 57 examples. Proteins 2014; 82:1101-1112. (c) 2013 Wiley Periodicals, Inc.

Succinate esters: odd-even effects in melting points

Dialkyl succinates show a pattern of alternating behavior in their melting points, as the number of C atoms in the alkane side chain increases, unlike in the dialkyl oxalates Joseph et al. (2011). Acta Cryst. B67, 525-534]. Dialkyl succinates with odd numbers of C atoms in the alkyl side chain show higher melting points than the immediately adjacent analogues with even numbers. The crystal structures and their molecular packing have been analyzed for a series of dialkyl succinates with 1 - 4 C atoms in the alkyl side chain. The energy difference (Delta E) between the optimized and observed molecular conformations, density, Kitaigorodskii packing index (KPI) and C-H center dot center dot center dot O interactions are considered to rationalize this behavior. In contrast to the dialkyl oxalates where a larger number of moderately strong C-H center dot center dot center dot O interactions were characteristic of oxalates with elevated melting points, here the molecular packing and the density play a major role in raising the melting point. On moving from oxalate to succinate esters the introduction of the C2 spacer adds two activated H atoms to the asymmetric unit, resulting in the formation of stronger C-H center dot center dot center dot O hydrogen bonds in all succinates. As a result the crystallinity of long-chain alkyl substituted esters improves enormously in the presence of hydrogen bonds from activated donors.

Hydrogelation of bile acid-peptide conjugates and in situ synthesis of silver and gold nanoparticles in the hydrogel matrix

Fabricating supramolecular hydrogels with embedded metal nanostructures is important for the design of novel hybrid nanocomposite materials for diverse applications such as biosensing and chemosensing platforms, catalytic and antibacterial functional materials etc. Supramolecular self-assembly of bile acid-dipeptide conjugates has led to the formation of new supramolecular hydrogels. Gelation of these molecules depends strongly on the hydrophobic character of the bile acids. The possibility of in situ fabrication of Ag and Au NPs in these supramolecular hydrogels by incorporating Ag+ and Au3+ salts was investigated via photoreduction. Chemical reductions of Ag+ and Au3+ salts in the hydrogels were performed without adding any external stabilizing agents. In this report we have shown that the color, size and shape of silver nanoparticles formed by photoreduction depend on the amino acid residue of the side chain.

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel -sheet in crystals

The crystal structure of a tripeptide Boc-Leu-Val-Ac(12)c-OMe (1) is determined, which incorporates a bulky 1-aminocyclododecane-1-carboxylic acid (Ac(12)c) side chain. The peptide adopts a semi-extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C-,C--dialkylated residue Ac(12)c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel -sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac(12)c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti-parallel -sheet in crystals, twisted or helical -sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted -sheet assembly in crystals. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd.

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel -sheet in crystals

The crystal structure of a tripeptide Boc-Leu-Val-Ac(12)c-OMe (1) is determined, which incorporates a bulky 1-aminocyclododecane-1-carboxylic acid (Ac(12)c) side chain. The peptide adopts a semi-extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C-,C--dialkylated residue Ac(12)c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel -sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac(12)c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti-parallel -sheet in crystals, twisted or helical -sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted -sheet assembly in crystals. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd.

QM/MM and Classical Molecular Dynamics Simulation of Histidine-Tagged Peptide Immobilization on Nickel Surface

The hybrid quantum mechanics (QM) and molecular mechanics (MM) method is employed to simulate the His-tagged peptide adsorption to ionized region of nickel surface. Based on the previous experiments, the peptide interaction with one Ni ion is considered. In the QM/MM calculation, the imidazoles on the side chain of the peptide and the metal ion with several neighboring water molecules are treated as QM part calculated by “GAMESS”, and the rest atoms are treated as MM part calculated by “TINKER”. The integrated molecular orbital/molecular mechanics (IMOMM) method is used to deal with theQMpart with the transitional metal. By using the QM/MM method, we optimize the structure of the synthetic peptide chelating with a Ni ion. Different chelate structures are considered. The geometry parameters of the QM subsystem we obtained by QM/MM calculation are consistent with the available experimental results. We also perform a classical molecular dynamics (MD) simulation with the experimental parameters for the synthetic peptide adsorption on a neutral Ni(1 0 0) surface. We find that half of the His-tags are almost parallel with the substrate, which enhance the binding strength. Peeling of the peptide from the Ni substrate is simulated in the aqueous solvent and in vacuum, respectively. The critical peeling forces in the two environments are obtained. The results show that the imidazole rings are attached to the substrate more tightly than other bases in this peptide.

QM/MM and Classical Molecular Dynamics Simulation of Histidine-Tagged Peptide Immobilization on Nickel Surface

The hybrid quantum mechanics (QM) and molecular mechanics (MM) method is employed to simulate the His-tagged peptide adsorption to ionized region of nickel surface. Based on the previous experiments, the peptide interaction with one Ni ion is considered. In the QM/MM calculation, the imidazoles on the side chain of the peptide and the metal ion with several neighboring water molecules are treated as QM part calculated by "GAMESS", and the rest atoms are treated as MM part calculated by "TINKER". The integrated molecular orbital/molecular mechanics (IMOMM) method is used to deal with the QM part with the transitional metal. By using the QM/MM method, we optimize the structure of the synthetic peptide chelating with a Ni ion. Different chelate structures are considered. The geometry parameters of the QM subsystem we obtained by QM/MM calculation are consistent with the available experimental results. We also perform a classical molecular dynamics (MD) simulation with the experimental parameters for the synthetic peptide adsorption on a neutral Ni(100) surface. We find that half of the His-tags are almost parallel with the substrate, which enhance the binding strength. Peeling of the peptide from the Ni substrate is simulated in the aqueous solvent and in vacuum, respectively. The critical peeling forces in the two environments are obtained. The results show that the in-tidazole rings are attached to the substrate more tightly than other bases in this peptide.

Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel member of the ATP-binding cassette (ABC) superfamily of membrane proteins. CFTR has two homologous halves, each consisting of six transmembrane spanning domains (TM) followed by a nucleotide binding fold, connected by a regulatory (R) domain. This thesis addresses the question of which domains are responsible for Cl^- selectivity, i.e., which domains line the channel pore.

To address this question, novel blockers of CFTR were characterized. CFTR was heterologously expressed in Xenopus oocytes to study the mechanism of block by two closely related arylaminobenzoates, diphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by both is voltage-dependent, with a binding site ≈ 40% through the electric field of the membrane. DPC and FFA can both reach their binding site from either side of the membrane to produce a flickering block of CFTR single channels. In addition, DPC block is influenced by Cl^- concentration, and DPC blocks with a bimolecular forward binding rate and a unimolecular dissociation rate. Therefore, DPC and FFA are open-channel blockers of CFTR, and a residue of CFTR whose mutation affects their binding must line the pore.

Screening of site-directed mutants for altered DPC binding affinity reveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6 abolishes most DPC block, greatly reduces single-channel conductance, and alters the direction of current rectification. Additional residues are found in TM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC block; other mutations move the DPC binding site from TM-6 to TM-12. The strengthened block and lower conductance due to mutation T1134F is quantitated at the single-channel level. The geometry of DPC and of the residues mutated suggest α-helical structures for TM-6 and TM-12. Evidence is presented that the effects of the mutations are due to direct side-chain interaction, and not to allosteric effects propagated through the protein. Mutations are also made in TM-11, including mutation S1118F, which gives voltage-dependent current relaxations. The results may guide future studies on permeation through ABC transporters and through other Cl^- channels.

Structural requirements for catalysis: studies on RTEM-1 β-lactamase

β-lactamases are a group of enzymes that confer resistance to penam and cephem antibiotics by hydrolysis of the β-lactam ring, thereby inactivating the antibiotic. Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Asp 132, a strictly conserved residue among the class A β-lactamases, appears to be involved in substrate binding, catalysis, or both. To study the contribution of residue 132 to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at position 132. Phenotypic screening of all mutants indicated that position 132 is very sensitive to amino acid changes, with only N132C, N132D, N132E, and N132Q showing any appreciable activity. Kinetic analysis of three of these mutants showed increases in K_M, along with substantial decreases in k_(cat). Efforts to trap a stable acyl-enzyme intermediate were unsuccessfuL These results indicate that residue 132 is involved in substrate binding, as well as catalysis, and supports the involvement of this residue in acylation as suggested by Strynadka et al.

Crystallographic and computer modeling studies of RTEM-1 β-lactamase have indicated that Lys 73 and Glu 166, two strictly conserved residues among the class A β-lactamases, appear to be involved in substrate binding, catalysis, or both. To study the contribution of these residues to β-lactamase function, site saturation mutagenesis was used to generate mutants coding for all 20 amino acids at positions 73 and 166. Then all 400 possible combinations of mutants were created by combinatorial mutagenesis. The colonies harboring the mutants were screened for growth in the presence of ampicillin. The competent colonys' DNA were sequenced, and kinetic parameters investigated. It was found that lysine is essential at position 73, and that position 166 only tolerated fairly conservative changes (Aspartic acid, Histidine, and Tyrosine). These functional mutants exhibited decreased kcat's, but K_M was close to wild-type levels. The results of the combinatorial mutagenesis experiments indicate that Lysis absolutely required for activity at position 73; no mutation at residue 166 can compensate for loss of the long side chain amine. The active mutants found--K73K/E166D, K73KIE166H, and K73KIE166Y were studied by kinetic analysis. These results reaffirmed the function of residue 166 as important in catalysis, specifically deacylation.

The identity of the residue responsible for enhancing the active site serine (Ser 70) in RTEM-1 β-lactamase has been disputed for some time. Recently, analysis of a crystal structure of RTEM-1 β-lactamase with covalently bound intermediate was published, and it was suggested that Lys 73, a strictly conserved residue among the class A β-lactamases, was acting as a general base, activating Ser 70. For this to be possible, the pK_a of Lys 73 would have to be depressed significantly. In an attempt to assay the pK_a of Lys 73, the mutation K73C was made. This mutant protein can be reacted with 2-bromoethylamine, and activity is restored to near wild type levels. ^(15)N-2-bromoethylamine hydrobromide and ^(13)C-2-bromoethylamine hydrobromide were synthesized. Reacting these compounds with the K73C mutant gives stable isotopic enrichment at residue 73 in the form of aminoethylcysteine, a lysine homologue. The pK_a of an amine can be determined by NMR titration, following the change in chemical shift of either the ^(15)N-amine nuclei or adjacent Be nuclei as pH is changed. Unfortunately, low protein solubility, along with probable label scrambling in the Be experiment, did not permit direct observation of either the ^(15)N or ^(13)C signals. Indirect detection experiments were used to observe the protons bonded directly to the ^(13)C atoms. Two NMR signals were seen, and their chemical shift change with pH variation was noted. The peak which was determined to correspond to the aminoethylcysteine residue shifted from 3.2 ppm down to 2.8 ppm over a pH range of 6.6 to 12.5. The pK_a of the amine at position 73 was determined to be ~10. This indicates that residue 73 does not function as a general base in the acylation step of the reaction. However the experimental measurement takes place in the absence of substrate. Since the enzyme undergoes conformational changes upon substrate binding, the measured pK_a of the free enzyme may not correspond to the pK_a of the enzyme substrate complex.