How do point amino acid substitutions affect the protein structure?

In order to estimate the influence of point mutations on the protein structure, 552 pairs of structurally aligned proteins with pairwise sequence identities greater than 60% were analyzed. We selected all isolated point mismatches from these alignments. The statistics of local conformational changes corresponding to these mismatches was studied. This study revealed two surprising aspects: (a) only an extremely minor fraction (< 3%) of all observed mutations leads to significant changes in local conformations; (b) observed substitutions, which affect local conformation are more frequently those of similar (high scoring) amino acid pairs rather than the substitutions thought of as "hazardous" (low scoring), although the overall average score of the changing substitutions is still lower than the corresponding average score for the substitutions that leave the structure unchanged.     

The crystal structure of an ‘All Locked’ nucleic acid duplex

‘Locked nucleic acids’ (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an ‘all LNA’ duplex containing exclusively modified β-d-2′-O-4′C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern.     

An amino acid packing code for α-helical structure and protein design

This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Delauney Tessellations to identify contacts, the knob-socket is a four-residue tetrahedral motif: a knob residue on one α-helix packs into the three-residue socket on another α-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the three-residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between two α-helices. Furthermore, the knob-socket model classifies three types of sockets: (1) free, favoring only intra-helical packing; (2) filled, favoring inter-helical interactions; and (3) non, disfavoring α-helical structure. The amino acid propensities in these three socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, we used a novel yet straightforward approach for the design of α-helical structure to validate the knob-socket model. Unique sequences for three peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no helix. These three peptides were synthesized, and helical content was assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure.     

The solution structure of the human retinoic acid receptor-β DNA-binding domain

Summary The three-dimensional structure of the DNA-binding domain of the human retinoic acid receptor- (hRAR-) has been determined by nuclear magnetic resonance spectroscopy in conjunction with distance geometry, restrained molecular dynamics and iterative relaxation matrix calculations. A total of 1244 distance restraints were obtained from NOE intensities, of which 448 were intra-residue and 796 inter-residue restraints. In addition 23 and 30 dihedral angle restraints were obtained from J-coupling data. The two zinc-finger regions of the 80-amino acid residue protein are followed by two -helices that cross each other perpendicularly. There is a short stretch of b-sheet near the N-terminus. The -helical core of the protein is well determined with a backbone root-mean-square deviation (r.m.s.d.) with respect to the average of 0.18 Å and 0.37 Å when the side chains of residues 31, 32, 36, 61, 62, 65 and 69 are included. The r.m.s.d. for the backbone of residues 5–80 is 0.76 Å. For the first finger (residues 8–28), the r.m.s.d. of the backbone is 0.79 Å. For the second finger (residues 44–62) the r.m.s.d. is 0.64 Å. The overall structure is similar to that of the corresponding domain of the glucocorticoid receptor, although the C-terminal part of the protein is different. The second -helix is two residues shorter and is followed by a well-defined region of extended backbone structure.     

The synthesis and structure of an n-terminal dodecanoic acid conjugate of α-conotoxin MII

Summary The α-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail,Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype α3β2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.     

Multiplicity of brain cysteine sulfinic acid decarboxylase — Purification,characterization and subunit structure

Cysteine sulfinic acid decarboxylase (CSAD), the rate-limiting enzyme in taurine biosynthesis, appears to be present in the brain in multiple isoforms. Two distinct forms of CSAD, referred to as CSAD I and CSAD II, were obtained on Sephadex G-100 column. CSAD I and CSAD II differ in (1) the elution profile on Sephadex G-100 column; (2) the sensitivity towards Mn²⁺, methione, and other sulfur-containing amino acids and (3) their immunologic properties. CSAD II has been purified to about 2,500-fold by a combination of column chromatographies and polyacrylamide gel electrophoresis (PAGE). The purity of the enzyme preparation was established as judged from the following observations: (1) a single protein band was observed under various electrophoretic conditions, e.g., 5–20% nondenaturing PAGE, 7% nondenaturing PAGE and 10% SDS-PAGE and (2) in nondenaturing PAGE, the protein band comigrated with CSAD activity. CSAD II has a molecular weight of 90 kDa and is a homodimer consisting of two 43 ± 2 kDa subunits. CSAD appears to require Mn²⁺ for its maximum activity. Other divalent cations fail to replace Mn²⁺ in activation of CSAD activity. However, the precise role of Mn²⁺ in the action of CSAD remains to be determined.     

Synthesis and structure–activity relationship of thiobarbituric acid derivatives as potent inhibitors of urease

A series of thiobarbituric acid derivatives 1–27 were synthesized and evaluated for their urease inhibitory potential. Exciting results were obtained from the screening of these compounds 1–27. Compounds 5, 7, 8, 11, 16, 17, 22, 23 and 24 showed excellent urease inhibition with IC₅₀ values 18.1 ± 0.52, 16.0 ± 0.45, 16.0 ± 0.22, 14.3 ± 0.27, 6.7 ± 0.27, 10.6 ± 0.17, 19.2 ± 0.29, 18.2 ± 0.76 and 1.61 ± 0.18 μM, respectively, much better than the standard urease inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM). Compound 3, 4, 10, and 26 exhibited comparable activities to the standard with IC₅₀ values 21.4 ± 1.04 and 21.5 ± 0.61μM, 22.8 ± 0.32, 25.2 ± 0.63, respectively. However the remaining compounds also showed prominent inhibitory potential The structure–activity relationship was established for these compounds. This study identified a novel class of urease inhibitors. The structures of all compounds were confirmed through spectroscopic techniques such as EI-MS and ¹H NMR.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Crystal structure determination of the β-cyclodextrin-p-aminobenzoic acid inclusion complex from powder X-ray diffraction data

The 1:1 inclusion complex of β-cyclodextrin and p-aminobenzoic acid was prepared and characterized by TG-DTA. The crystal structure of the complex was solved directly from powder X-ray diffraction data using the direct space approach and refined using Rietveld refinement techniques. The complex crystallizes in monoclinic P2₁ space group, with unit cell parameters a = 20.7890 ?, b = 10.2084 ?, c = 15.1091 ?, β = 110.825°, V = 2997 ?³. The amino group is located at the wide side of the β-cyclodextrin cavity, forming hydrogen bonds with β-cyclodextrin, and the carboxyl group is located at the narrow side. The crystallographic data obtained from powder diffraction data were compared with the single crystallographic data, and the result shows that solving crystal structure of cyclodextrins inclusion complexes of such complexity is accessible to powder diffractionists to some extent.     

Peptide nucleic acid (PNA): A model structure for the primordial genetic material?

It is proposed that the primordial genetic material could have been peptide nucleic aicds,i.e., DNA analogues having a peptide backbone. PNA momomers based on the amino acid, , -diaminobutyric acid or ornithine are suggested as compounds that could have been formed in the prebiotic soup. Finally, the possibility of a PNA/RNA world is presented, in which PNA constitutes the stable genetic material, while RNA which may be polymerized using the PNA as template accounts for enzymatic activities including PNA replication.     

Synthesis and structure–activity relationship analysis of caffeic acid amides as selective matrix metalloproteinase inhibitors

Four series of acid amides were synthesized, and through measurement using a fluorogenic substrate assay with human recombinant MMP-1, MMP-2 and MMP-9, compound 3f showed considerable inhibitory activities against MMP-2, MMP-9 and the best selectivity over MMP-1. Preliminary structure–activity relationship analysis indicated that caffeic acid amides with electron-donating groups at para-position of amino phenyl group showed better inhibitory activities and selectivity than those with electron-withdrawing groups, and the presence of adjacent dihydroxy in the caffeoyl group was very important for the MMP-2 and MMP-9 inhibitory activities.     

Quantum-chemical investigation of the structure and the antioxidant properties of α-lipoic acid and its metabolites

Quantum-chemical computations were used to investigate the structure-antioxidant parameter relationships of α-lipoic acid and its natural metabolites bisnorlipoic acid and tetranorlipoic acid in their oxidized and reduced forms. The enantiomers of lipoic and dihydrolipoic acid were optimized using the B3LYP/6-311+G(3df,2p), B3LYP/aug-cc-pVDZ and MP2(full)/6-31+G(d,p) levels of theory as isolated molecules and in the presence of water. The geometries of the metabolites and the values of their antioxidant parameters (proton affinity, bond dissociation enthalpy, adiabatic ionization potential, spin density, and the highest occupied molecular orbital energy) were calculated at the B3LYP/6-311+G(3df,2p) level of theory. The results obtained reveal similarities between these structures: a pentatomic, nonaromatic ring is present in the oxidized forms, while an unbranched aliphatic chain (as found in saturated fatty acids) is present in both the oxidized and the reduced forms. Analysis of the spin density and the highest occupied molecular orbital energy revealed that the SH groups exhibited the greatest electron-donating activities. The values obtained for the proton affinity, bond dissociation enthalpy and adiabatic ionization potential indicate that the preferred antioxidant mechanisms for α-lipoic acid and its metabolites are sequential proton loss electron transfer in polar media and hydrogen atom transfer in vacuum.     

δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.     

The synthesis and structure–activity relationship of substituted N-phenyl anthranilic acid analogs as amyloid aggregation inhibitors

It is believed that β-amyloid aggregation is an important event in the development of Alzheimer’s disease. In the course of our studies to identify β-amyloid aggregation inhibitors, a series of N-phenyl anthranilic acid analogs were synthesized and studied for β-amyloid inhibition activity. The synthesis, structure–activity relationship, and in vivo activity of these analogs are discussed.     

Zinc complexes with 1,2,4-triazole functionalized amino acid derivatives: Synthesis, structure and β-lactamase assay

Coordinating abilities of 4R-1,2,4-triazole derivatives (R = glycine ethyl ester (L1), glycine (L2), diethylamino malonate (L3), methionine (L4) and diethyl aminomethylphosphonate (L5)) towards Zn^II ions have been studied in solution, in solid state and versus three zinc-β-lactamases. The crystal structure of [Zn₃(L4)₆(H₂O)₆] (6) is described; it is the first crystal structure involving a 1,2,4-triazole functionalized methionine. It forms a trinuclear complex with central zinc octahedrally coordinated by only L4, whereas terminal zinc ions coordination sphere is completed by three water molecules. L4 exhibits a dual functionality of a bridging bidentate ligand as well as an anion. A dense hydrogen bonding network connects these trinuclear entity into a 3D supramolecular network. The Zn^II ions in 6 are held at equidistance (3.848 Å) which coincidently matches with the corresponding Zn?Zn distance in the binuclear zinc enzyme from Bacillus cereus (3.848 and 4.365 Å). Among L1-L5 screened for β-lactamase assay, L4 shows modest inhibition for BcII enzyme.     

ω3 fatty acid desaturases from microorganisms: structure, function, evolution, and biotechnological use

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.