Single crystals of a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine

Lamellar single crystals were formed from a random copolypeptide composed of γ-benzyl l-glutamate and l-phenylalanine at the ratio of 4 to 1. The copolypeptide takes the αhelical structure. The crystals were formed by casting dilute solutions at room temperature from a solvent consisting of a 1 to 1 mixture of chloroform and trifluoroacetic acid and were observed by electron microscopy. The average crystal thickness was 670 a in the as-polymerized sample, and 580 a in a fractionated sample. The thickness was decreased by annealing at temperatures above 110 C. A hexagonal form, a group of three orthorhombic forms (group 1), and a group of an orthorhombic form and two monoclinic forms (group II) were observed by electron diffraction. The diversity of the crystal structures is suggested to be caused by a variation in crystallization conditions during evaporation of the solvent. The hexagonal form and the structures of group I are changed into the structures of group II by annealing. The crystal structures other than the hexagonal form indicate on ordered arrangements of side chains in the crystals.     

Solid state properties of anomeric 1-O-n-octyl-d-glucopyranosides

The anomers of 1-O-n-octyl-D-glucopyranosides exhibit different crystal packing and thermodynamic properties. Crystallization either from solution or by epitaxy of the α-anomer resembles that of other amphiphiles, such as lysolecithin, and is isostructural to the decyl homologue. The β-anomer crystallizes into a unique form, independent of conditions, with the longest cyrstallographic axis parallel to the best developed crystal face. Both compounds exhibit two phase transitions, one near 70°C, the other above 100°C. The latter corresponds to melting to an isotropic liquid for both forms, but the former is distinctly different for the two anomers. Thus, birefringence is lost only with the β-anomer, while the enthalpy change is two-fold larger for the α-anomer. The crystal packing of the two compounds are thus clearly different.     

Crystal structure of a tripeptide,L-alanyl-glycyl-glycine and its relevance to the poly(glycine)-II type of conformation

A tripeptide molecule, L -alanyl-glycyl-glycine, crystallizes in the form of a left-handed helix with (?,ψ) = ?83°, 170°. A pseudohexagonal packing arrangement and interchain hydrogen-bonded interactions are reminiscent of the model for the structure of poly(glycine)-II. Observations of certain intermolecular interactions appear to be relevant to the stereochemical assumptions incorporated in the models proposed for poly(glycine)-II and related polypeptides.     

Single crystals of poly-L-lysine

Crystals of poly-L -lysine have been grown from aqueous solution in the presence of divalent anions. The most stable of these incorporate the HPO ion and are precipitated by the addition of sodium monohydrogen phosphate to solutions of poly-L -lysine HBr. Precipitation at or slightly above room temperature gives rise to single crystals of α-poly-L -lysine HPO₄ in the form of hexagonal lamellae about 150 Å thick. The axes of the helical polypeptide molecules are oriented normal to the planes of the lamellae, and since molecular length is about 1100 Å in the α-helical conformation, these helices must be folded. The a parameter of the hexagonal unit cell is 19.55 Å for crystals immersed in mother liquor, and the lysine side chains are almost fully extended. Precipitation brought about by heating the same solutions to about 75°C produces micro-crystals of β-poly-L -lysine HPO₄. A mode of packing of the anions in these crystals is proposed tentatively on the basis of an intersheet spacing determined from x-ray powder diffraction patterns. In general, α crystals are transformed to β structures on drying; conditions under which the transition can either be forestalled or reversed are discussed.     

Biochemical and hydrolytic properties of multiple thermostable α-galactosidases from Streptomyces griseoloalbus: Obvious existence of a novel galactose-tolerant enzyme

The multiple α-galactosidases from Streptomyces griseoloalbus—α-Gal I, α-Gal II and α-Gal III were purified to homogeneity by a two-step chromatographic process. The molecular masses and pI of the three enzymes were 72, 57 and 35 kDa, and 4.41, 5.6 and 6.13, respectively. α-Gal I showed N-terminal sequence homology to S. coelicolor A3(2) family 27 α-galactosidase. The optimum pH and temperature of the three α-galactosidases were 5.0, 6.5 and 5.5 and 65, 50 and 55 °C, respectively. α-Gal I was stable up to 65 °C and α-Gal II and α-Gal III up to 55 °C for 2 h. Based on the hydrolytic properties α-Gal I could be classified as a member of GH27 family and α-Gal II and α-Gal III as members of GH36 family. Metal cations like Hg²⁺, Ag²⁺ and Cu²⁺ inhibited enzyme activity while Mg²⁺ enhanced the activity of α-Gal I. Interestingly α-Gal I showed unusual tolerance to even higher concentrations of galactose, unlike the other two α-galactosidases, which were competitively inhibited by galactose. Melibiose was a competitive inhibitor of all three enzymes. Histidine, tryptophan and carboxylic residues were essential for catalytic action of the three α-galactosidases.     

Laser Raman studies of conformational variations of poly-L-lysine

The frequencies and intensities of the laser Raman spectra of poly-L -lysine (PLL) have been observed in the following studies: (1) the thermally induced α-to-β transition which occurs with increasing temperature at high pH; (2) the ionized form to α transition at 10°C by increasing pH; and (3) the ionized form to α transition by ionic strength at low pH. The frequency-dependent bands which have been observed are the amide I (in H₂O), amide I′ (in D₂O), amide III, and C–C stretch. It has been found possible to assign an unique set of frequencies and intensities to each conformation of PLL of α, β, and ionized form. In this way the nature of the conformations intermediate in the transitions can be determined. The frequencies of the amide III and amide III′ are very weak in the α-helix and somewhat higher than usual in the β form. Hence it appears the amide III and amide III′ bands may differ from one type of polypeptide to another with the same backbone conformation.     

β-Turn conformations in crystal structures of model peptides containing α,α-Di-n-propylglycine and α,α-Di-n-butylglycine

The crystal state conformations of three peptides containing the α,α-dialkylated residues. α,α-di-n-propylglycine (Dpg) and α,α-di-n-butylglycine (Dbg), have been established by x-ray diffraction. Boc-Ala-Dpg-Alu-OMe (I) and Boc-Ala-Dbg-Ala-OMe (III) adopt distorted type II β-turn conformations with Ala (1) and Dpg/Dbg (2) as the corner residues. In both peptides the conformational angles at the Dxg residue (I: ? = 66.2°, ψ = 19.3°; III: ? = 66.5°. ψ = 21.1°) deviate appreciably from ideal values for the i + 2 residue in a type II β-turn. In both peptides the observed (N…O) distances between the Boc CO and Ala (3) NH groups are far too long (1: 3.44 Å: III: 3.63 Å) for an intramolecular 4 → 1 hydrogen bond. Boc-Ala-Dpg-Ata-NHMe (II) crystallizes with two independent molecules in the asymmetric unit. Both molecules HA and HB adopt consecutive β-turn (type III-III in HA and type III-I in IIB) or incipient 3₁₀-helical structures, stabilized by two intramolecular 4 → 1 hydrogen bonds. In all four molecules the bond angle N-C^α-C′ (τ) at the Dxg residues are ≥ 110°. The observation of conformational angles in the helical region of ?,ψ space at these residues is consistent with theoretical predictions. © 1995 John Wiley & Sons, Inc.     

Crystals of modified fibrinogen: Size,shape and packing of molecules

We have produced several new macroscopic crystal forms and a variety of microcrystals from modified flbrinogens. Bovine and rabbit flbrinogens crystallize after limited digestion by a bacterial protease or α-chymotrypsin. The fibrinogens making up these crystals are largely intact and highly clottable. Tentative molecular packing arrangements for two crystal forms have been deduced. The crystal morphology and cleavage planes were used in this analysis. The characteristic α-helical coiled-coil reflections arid spikes of intensity in certain directions in the crystal X-ray patterns serve as markers for the orientation of the fibrinogen molecules. Changes that occur in one of the forms during preparation for electron microscopy, as shown by comparison with X-ray experiments on crystals in various stains and solvents, support this packing model. These studies provide preliminary evidence that fibrinogen is about 450 Å in length and that the molecules bond end-to-end to form filaments making up the crystals.     

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High‐density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α‐helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule‐like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA‐I, shows relatively large volume contraction, ΔV_unf = ?0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα_unf ≤ 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α‐helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA‐I shows large thermal expansion coefficient, α(5°) = 15·10^?4 K^?1, that rapidly declines upon heating from 5 to 40°C, α(40°) ? α(5°) = ?4·10^?4 K^?1; apolipoprotein C‐I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein–lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA‐I or apoC‐I, dimyristoyl phosphatidylcholine, and 0–20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.     

General model of myosin filament structure. 3. Molecular packing arrangements in myosin filaments

Following the original proposals about myosin filament structure put forward as part of a general myosin filament model (Squire, 1971, 1972) it is here shown what the most likely molecular packing arrangements within the backbones of certain myosin filaments would be assuming that the model is correct. That this is so is already indicated by recently published experimental results which have confirmed several predictions of the model (Bullard and Reedy, 1972; Reedy et al., 1972; Tregear and Squire, 1973).The starting point in the analysis of the myosin packing arrangements is the model for the myosin ribbons in vertebrate smooth muscle proposed by Small &; Squire (1972). It is shown that there is only one reasonable type of packing arrangement for the rod portions of the myosin molecules which will account for the known structure of the ribbons and which is consistent with the known properties of myosin molecules. The dominant interactions in this packing scheme are between parallel myosin molecules which are related by axial shifts of 430 Å and 720 Å. In this analysis the myosin rods are treated as uniform rods of electron density and only the general features of two-strand coiled-coil molecules are considered.Since the general myosin filament model is based on the assumption that the structures of different types of myosin filament must be closely related, the packing scheme derived for the myosin ribbons is used to deduce the structures of the main parts (excluding the bare zones) of the myosin filaments in a variety of muscles. It is shown in each case that there is only one packing scheme consistent with all the available data on these filaments and that in each filament type exactly the same interactions between myosin rods are involved. In other words the myosin-myosin interactions involved in filament formation are specific, they involve molecular shifts of either 430 Å or 720 Å, and are virtually identical in all the different myosin filaments which have been considered. Apart from the myosin ribbons, these are the filaments in vertebrate skeletal muscle, insect flight muscle and certain molluscan muscles.In the case of the thick filaments in vertebrate skeletal muscle the form of the myosin packing arrangement in the bare zone is considered and a packing scheme proposed which involves antiparallel overlaps between myosin rods of 1300 Å and 430 Å. It is shown that this scheme readily explains the triangular profiles of the myosin filaments in the bare zone (Pepe, 1967, 1971) and many other observations on the form of these myosin filaments.Finally it is shown that the cores of several different myosin filaments, assuming they contain protein, may consist of different arrangements of one or other of two types of core subfilament.     

Acyl chain interdigitation in saturated mixed-chain phosphatidylcholine bilayer dispersions

The molecular packing of various fully hydrated mixed-chain phosphatidylcholines was studied by X-ray diffraction and electron microscopy. All of the mixed-chain phosphatidylcholines under study were shown to adopt a lamellar or bilayer form in aqueous media. The bilayer thickness of these mixed-chain phosphatidylcholines was determined from the lamellar repeat distance in the small-anglé X-ray diffraction region by controlled swelling experiments. At T greater than Tm, the bilayer thickness of C(18):C(12)PC and C(18):C-(10)PC is found to be comparable to that of C(14):C(14)PC. In contrast, the bilayer thickness of these highly asymmetric phosphatidylcholines is considerably less than that of the symmetric C(14):C(14)PC at temperatures below Tm. Moreover, the wide-angle X-ray diffraction patterns taken at T less than Tm consist of at least two sharp reflections at 4.2 and 4.6 A. These X-ray diffraction data suggest that these highly asymmetric mixed-chain phospholipids, in excess water, form mixed interdigitated bilayers in the gel state and that the acyl chain packing in the gel-state bilayer is not hexagonal. The freeze-fracture planes of these mixed-chain phosphatidylcholines are discontinuous at T less than Tm, supporting the conclusion drawn from X-ray diffraction data that these highly asymmetric phosphatidylcholines form interdigitated bilayers at temperatures below Tm. The molecular packing of fully hydrated C(18):C(14)PCs in bilayers is distinctively different from that of C(18):C(10)PCs or C(18):C(10)PCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

A molecular dynamics study of the pentacyclo-undecane (PCU) cage polypeptides of the type Ac-3Ala-Cage-3Ala-NHMe

An extensive exploration of the conformational space of the seven-residue peptide sequences, Ac-Ala-Ala-Ala-Cage-Ala-Ala-Ala-NHMe and the model peptide Ac-Ala-Ala-Ala-Ala-Ala-Ala-Ala-NHMe, was carried out using single trajectories of molecular dynamics (MD) in the solution phase using the periodic boundary conditions. Our MD studies revealed that the majority of the motifs of the PCU cage peptide exist as type I–III β-turns along with their mirror conformations, viz. type I′–III′ β-turns. This peptide sequence adopted a U-shaped backbone, with alpha-helical characteristics. The results reported here provide further evidence that the PCU cage amino acid exhibits C_7eq, C_7aq, α_R and α_L conformations in aqueous solution.     

ATP Content and Sperm Motility of Extended Bovine Semen under Different Storage Conditions

The effects of incubation temperature (+20°C vs +35°C) and media type on the ATP content and motility of spermatozoa were determined in fresh bovine semen in order to develop a method for assaying post-thaw quality. Semen was obtained from 3 bulls at 2 occasions. The spermatozoa were washed using a Ficoll-containing medium before being resuspended in each of 4 different media (I. 0.9 % NaCl; II. Trisbuffer solution; III. seminal plasma; IV. seminal plasma + Tris-buffer solution) and incubated for 6 h. The least-squares means for ATP content were higher (p ≤ 0.05) at +20°C than +35°C for all media except no. I. By contrast, the least-squares means for sperm motility were higher (p ≤ 0.05) at +35°C than at +20°C in media II and III. A decrease over time in ATP content and motility at both temperatures was also observed. The single most important factor responsible for changes in ATP content and sperm motility was the temperature and the medium, respectively.     

Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form.

The crystal structure of the NS3 protease of the hepatitis C virus (BK strain) has been determined in the space group P6(3)22 to a resolution of 2.2 A. This protease is bound with a 14-mer peptide representing the central region of the NS4A protein. There are two molecules of the NS3(1-180)-NS4A(21'-34') complex per asymmetric unit. Each displays a familiar chymotrypsin-like fold that includes two beta-barrel domains and four short alpha-helices. The catalytic triad (Ser-139, His-57, and Asp-81) is located in the crevice between the beta-barrel domains. The NS4A peptide forms an almost completely enclosed peptide surface association with the protease. In contrast to the reported H strain complex of NS3 protease-NS4A peptide in a trigonal crystal form (Kim JL et al., 1996, Cell 87:343-355), the N-terminus of the NS3 protease is well-ordered in both molecules in the asymmetric unit of our hexagonal crystal form. The folding of the N-terminal region of the NS3 protease is due to the formation of a three-helix bundle as a result of crystal packing. When compared with the unbound structure (Love RA et al., 1996, Cell 87:331-342), the binding of the NS4A peptide leads to the ordering of the N-terminal 28 residues of the NS3 protease into a beta-strand and an alpha-helix and also causes local rearrangements important for a catalytically favorable conformation at the active site. Our analysis provides experimental support for the proposal that binding of an NS4A-mimicking peptide, which increases catalytic rates, is necessary but not sufficient for formation of a well-ordered, compact and, hence, highly active protease molecule.     

X-ray diffraction studies on the structure of hydrated collagen

The temperature dependence of the humidity-sensitive spacing, d, related to the lateral packing of collagen molecules was measured for fully hydrated collagen. In the vicinity of 0°C, a sudden change in d was observed, which was reversible with temperature. In the diffraction profile, below 0°C, a set of diffraction peaks identified with the hexagonal crystalline form of ice was observed. With the reduction in water content, the intensity of the set of diffraction peaks decreased and was found to be zero at a water content of 0.38 g/g collagen. These results were considered to be caused by the frozen water in collagen fibril below 0°C. According to the water content dependence of d, it was considered that up to a certain water content water absorbed would be stowed in the intermolecular space of collagen and above that water content water molecules would aggregate to make pools, i. e., extrafibrillar spaces. The unfreezable bound water was considered to be located in the intermolecular space of collagen. Size of the extrafibrillar space, determined from the intensity analysis of a smallangle x-ray scattering pattern, corroborates the speculation that the water showed in the extrafibrillar space is freezable and free. The formation of the hexagonal crystalline form of ice in the extrafibrillar space was considered to cause the sudden change in d at 0°C.     

The Effects of 1,2-Diacylglycerols on the Structural Organization of Model Membranes and Their Fusion

Abstract

The effects of synthetic and natural 1,2 diacylglycerols (DAG) on structural organization of lamellar dispersions of phosphatidylcholine (PC) or PC: phosphatidylinositol (PI) mixtures has been studied with the help of NMR spectroscopy. Asymmetric accumulation of natural DAG in two-component model membranes was achieved by their treatment with phospholipase C specific for PI. It was found that high concentrations (20 mol per cent) of synthetic DAG are needed to induce partial lipid bilayer transition from lamellar into hexagonal and/or isotropic phase. Asymmetric accumulation of natural DAG in thionphosphatidylcholine: PI bilayers treated with phospholipase C resulted in modification of bilayer packing at DAG concentrations as low as 1.5 mol per cent and at physiological temperature (37°C). With the help of fluorescence spectroscopy it was shown that formation of DAG in one of membrane layers of large mono-bilayer liposomes results in the membranes destabilization and fusion.     

Thermotropic mesomorphism of cholesteryl myristate. An electron diffraction study

Electron diffraction measurements on heated or cooled microcrystals of cholesteryl myristate, which are grown from solution or epitaxially, on benzoic acid, provide further structural information about its mesomorphic behavior. At subambient temperatures (less than -65 degrees C), a new crystal form is observed which doubles the unit cell axes in the (001) plane. At the major crystalline in equilibrium with smectic endotherm at 70 degrees C, evidence is found for the existence of a pretransition crystal packing. The smectic phase, which coexists with this pretransition crystal form, is composed of relatively well-ordered layers, probably with a monolayer-type packing. Cooling the cholesteric phase to the crystalline form causes a rotational disorder which is not yet understood.     

Sequence dependence of the B to Z transition in crystals and aqueous NaCl solutions for deoxyoligonucleotides containing all four canonical DNA bases

A laser Raman study has been made on the conformation of a series of self-complementary octameric deoxynucleotides that contain all four canonical deoxynucleotide bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] in order to determine which sequences will crystallize in the Z form and which sequences will go into the Z form in aqueous solution at high salt concentrations (4-6 M NaCl). All four octadeoxynucleotides, d(TGCGCGCA) (I), d(CACGCGTG) (II), d(CGTGCACG) (III), and d(CGCATGCG) (IV), have been crystallized from low-salt solutions. The Raman spectra of microcrystals show that I, II, and IV crystallize in a rigorous Z form while III crystallizes in the B form. Sequences I and II go into a Z form in 4-6 M NaCl solution at 0 degrees C while sequences III and IV remain in the B form in 6 M salt. There are substantial differences in the Raman spectra of oligonucleotides in the Z form found in the crystal and in high-salt solutions. The Raman spectra of the Z forms in 6 M NaCl solution at 0 degrees C are not linear combinations of the Raman spectra of the complete Z form in the crystal and the complete B form in low-salt solutions. The terminal residues of these oligomers do not appear to be in a strict Z form. A detailed analysis of the ring puckers and syn/anti conformation for all of the residues both in solution and in the crystal has been made.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Some Properties of Acid-stable α-Amylases from Shochu koji (Aspergillus kawachii)

Aspergillus kawachii α-amylase [EC 3.2.1.1] I and II were purified from shochu koji extract by DEAE Bio-Gel A ion exchange chromatography, Sephacryl S-300 gel chromatography (pH 3.6), coamino dodecyl agarose column chromatography and Sephacryl S-200 gel chromatography. By gel chromatography on a Sephacryl S-300 column, the molecular weights of the purified α-amylase I and II were estimated to be 104,000 and 66,000, respectively. The isoelectric points of α-amylase I and II were 4.25 and 4.20, respectively. The optimal pH range of α-amylase I was 4.0 to 5.0, and the optimum pH of α-amylase II was 5.0. The optimum temperatures of both α-amylases were around 70°C at pH 5.0. Both α-amylases were stable from pH 2.5 to 6.0 and up to 55°C, retaining more than 90% of the original activities. Heavy metal ions such as Hg^{2 +} and Pb^{2 +} were potent inhibitors for both α-amylases.