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PubMed1978

The covalent structure of bovine liver rhodanese. Isolation and partial structural analysis of cyanogen bromide fragements and the complete sequence of the enzyme.

Cyanogen bromide fragments from reduced and carboxymethylated rhodanese have been isolated by gel filtration and ion exchange chromatography on columns of Sephadex G-50 and sulfoethyl-Sephadex C-25, respectively. Partial or complete structural analysis of these fragments has permitted the ordering in sequence of all eleven of the nonaligned tryptic peptides from citraconylated, S-carboxymethylcysteinyl-rhodanese and has thus provided the complete covalent structure of the enzyme. Rhodanese is a single polypeptide of 293 residues and the molecule weight calculated from the covalent structural analysis is about 32,900. The cysteinyl residue implicated in the catalytic function of rhodanese is at position 247. In some preparations of the enzyme the NH2-terminal dipeptide Val-His is missing and the sequence begins with the glutamine at position 3. The rhodanese thus obtained contains 291 amino acid residues and possesses full enzymic activity. X-ray crystallographic analysis of rhodanese has shown that the halves of the molecule (Domains I and II) are nearly identical in conformation. Comparative analysis of the sequences in Domains I and II containing residues with conformationally equivalent alpha C atoms has revealed some degree of homology between the halves of the rhodanese polypeptide. Nethertheless, the structural equivalence of the rhodanese domains is reflected much more by their similarity in tertiary structural than by their sequence homology, even when the sequence comparisons are optimized with reference to the crystallographic results.

Amino Acid Sequence↗

PubMed1988

A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.

A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.

Carbohydrate Conformation↗

PubMed1987

Electron diffraction structure analysis of phospholipids.

The use of lamellar electron diffraction data from epitaxially oriented phospholipid crystals for quantitative structure analysis is described in this review of the technique. It is seen that an appropriate correction for crystal texture, which is justified by the analysis of low-dose lattice images, enables these intensity data to be used much as they would be used in X-ray crystallography. Analyses of two classes of phospholipids are reviewed, revealing that the recently determined lamellar structures of ether-linked phosphatidylethanolamine and phosphatidylcholine and quite similar to the acyl-chain structures. Preliminary analysis of 1,2-dihexadecyl-sn-glycerophospho-N-methyl ethanolamine indicates that the headgroup conformation may be similar to that of the ether-linked lecithin.

Microscopy, Electron↗

PubMed1990

In vitro secondary structure analysis of mRNA from lacZ translation initiation mutants.

mRNA secondary structure can be an important determinant of the efficiency of translation initiation. To study the effect of secondary structure on translation initiation, in vitro secondary structure analysis was performed on 32 lacZ RNA transcripts that differ in their in vivo translation initiation efficiencies because of mutations. We have shown that well-translated RNA has a relatively unstructured translation initiation region in vitro. In contrast, the translation initiation region of many of the poorly translated RNA transcripts is involved in a stem-loop structure. Mutations that decrease the in vitro stability of the stem-loop increase the frequency of translation initiation. The sequences responsible for forming this stem-loop structure were localized to a small region of RNA. The results confirm some of the previous predictions of the RNA secondary structure of the mutant RNAs based on computer modeling, but they disagree with some of the predicted long-range interactions.

Base Composition↗

PubMed1991

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.

Adenosine Diphosphate Ribose↗

PubMed1988

Structure analysis of fibrinogen by electron microscopy and image processing.

Human fibrinogen was observed by electron microscopy following rotary shadowing with tungsten. Structure analysis of the molecules was performed by image processing of electron micrographs. A method is described for selection, alignment, and classification of molecules. The widely accepted overall trinodular structure of the protein was observed. The flexibility about the central domain of the molecule was quantitatively analyzed. A Gaussian distribution of this conformational parameter was obtained having an average corresponding to a maximally extended structure. Correspondence analysis applied to the aligned images showed that the degree of folding of the molecule was continuously distributed. The averaged structure of fibrinogen was estimated to be 450 A long. The central domain had a diameter of 50 A and the peripheral domains were 90 A long and 50 A wide. The latter regions had two separated maxima of scattering density.

Fibrinogen↗

PubMed1992

Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis.

An X-ray structure analysis of a crystal of mutant Asp229----Ala of cyclodextrin glycosyltransferase from Bacillus circulans (Ec 2.4.1.19) that had been shortly exposed to beta-cyclodextrin showed density corresponding to a maltose bound at the catalytic center. The crystal structure was refined to an R-factor of 18.7% at 2.5-A resolution. The catalytic center is defined by homology with the structurally known alpha-amylases and by the observation that mutants Asp229----Ala and Asp328----Ala are almost inactive. By model building, the density-defined maltose was extended to a full beta-cyclodextrin, which then indicated the general locations of seven subsites for glucosyl units. The catalytically competent residues Asp229, Glu257, and Asp328 are at the reducing end of the density-defined maltose. In the unligated wild-type structure, Glu257 and Asp328 form a 2.6-A hydrogen bond between their carboxylates in an arrangement that resembles those of the catalytically competent carboxylates in acid proteases. Presumably, the first catalytic step is an attack of the proton between Glu257 and Asp328 on the oxygen of the glycosidic bond.

Bacillus↗

PubMed1985

Structural analysis of hepatitis B surface antigen by monoclonal antibodies.

A method has been developed for the analysis of hepatitis B surface antigen (HBsAg) antigenic structure at the molecular level that creates "fingerprints" or "signatures" of various hepatitis B viral (HBV) strains. This technique employs high affinity IgM and IgG monoclonal antibodies (anti-HBs) directed against distinct and separate determinants on HBsAg. In performing this antigenic structural analysis, separate binding curves for different monoclonal anti-HBs are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum by radioimmunoassay. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples are aligned by an iterative least-squares procedure to generate the numerical signature characteristic of the viral strain. The numerical signatures are then displayed on a computer-graphic plot. The signature profiles of HBsAg subtypes are a true reflection of their antigenic structure, and in vertical and horizontal transmission studies the molecular characteristics of the viral epitopes are conserved. By signature analysis we found substantial antigenic heterogeneity among the ayw3 strain both in the U.S. and France, as well as in populations of the Far East and Africa. Populations in Ethiopia, Gambia, and the Philippines were infected with two antigenically distinct HBV strains. In some newly identified HBV strains, it was found that epitopes identified by some monoclonal antibodies were absent or substantially reduced, which suggested that a genetic mutation may have occurred. Thus this study suggests that there is far more antigenic heterogeneity in HBV than previously recognized. These variants are antigenically distinct from each other at the epitope level, and were heretofore unrecognized by polyvalent anti-HBsAg antibodies.

Adult↗

PubMed1991

Primary structure analysis of polypeptides.

Primary structure determination together with other techniques such as amino acid analysis, peptide mapping, and polyacrylamide gel electrophoresis provide a physical characterisation of a polypeptide. This has been valuable in the research field for some time, but is now being applied to problems which arise in the areas of process development, formulation and QA of protein products. Probably the most significant contribution is elucidation of processing events, since this may go undetected by other techniques. Thus, with the emergence of various protein products from biotechnology, a technique which has been of great value in research activities for many years is finding its place in the range of techniques used for process development, formulation and QA.

Amino Acid Sequence↗

PubMed1991

[Structure analysis of the skin surface using computer-assisted laser profilometry. New method for the quantitative assessment of roughness structure of the skin].

Up to now, it has been difficult to record the roughness of the skin and to define it satisfactorily by measuring techniques. A new method for computer-assisted structural analysis of the skin surface is introduced, which uses laser beams for measurement without contact. This measuring technique is suitable for quantitative characterization of normal skin surfaces and for pathological alterations to the skin surface. Because of the dynamic properties of the skin, it is characterized by means of silicone replicas of the skin surface. Different parameters of roughness are determined and completed by mathematical and statistical processes such as Fourier transform and an autocorrelation function. These processes are related to the digitally stored three-dimensional profile of the skin surface. The new method is presented by means of some examples characterizing a typical clinical finding of rough eczematous skin. A comparison with photographs taken with a scanning electron microscope demonstrates the quality of resolution achieved with the measuring technique.

Fourier Analysis↗

PubMed1989

Molecular cloning, nucleotide sequence and fine-structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose-1,6-biphosphate aldolase to class I and class II aldolases.

The Corynebacterium glutamicum fda gene encoding fructose-1,6-biphosphate (FBP) aldolase has been isolated by complementation of an Escherichia coli mutant. The nucleotide sequence of a 3371 bp chromosomal fragment containing the C. glutamicum fda gene was determined. The N-terminal amino acid sequence of C. glutamicum FBP aldolase identified the correct initiation site for the fda gene, and a molecular weight of 37,092 was predicted for the fda polypeptide. S1 nuclease mapping identified the transcriptional start site, and Northern hybridization analysis indicated that the fda gene encodes a single 1.3 kb transcript. The primary structure of C. glutamicum FBP aldolase shows strong homology to class II FBP aldolases. Conservation of primary structure was observed between class I and class II aldolases, but several residues essential for catalytic activity in class I aldolases were absent from class II aldolases.

Amino Acid Sequence↗

PubMed1989

Highlights of protein structural analysis.

This article describes highlights of the state of the art in protein structural analysis, and comments on the current trends toward increased sensitivity and integrated isolation-structure methodologies.

Amino Acids↗

PubMed1990

Structural analysis of mitochondrial pores.

Structural information about the channel in the mitochondrial outer membrane, derived from sequence analysis and electron microscopy of two-dimensional crystals, is summarized. A model for the channel is presented, consisting of a cylindrical beta-barrel that is formed by one or two 30-kDa polypeptides, with an alpha-carbon backbone diameter of 3.8 nm. The radial distributions of basic amino acids and lipid-contact regions on the projected cylinder are mapped relative to interchannel bonding sites inferred from channel packing in the arrays. Speculation on the kinds of conformational changes that the channel might undergo is also presented.

Amino Acid Sequence↗

PubMed1978

Fine structure analysis of the threonine operon in Escherichia coli K-12.

A fine structure analysis of the threonine operon in Escherichia coli K-12 was performed by deletion mapping. Lambda transducing bacteriophages carrying various parts of the threonine operon were isolated from strains in which the lacZ gene was fused to a thr gene. We tested for recombination between deletions of the threonine promotor extending into the threonine operon, carried by the phage, and bacterial thr auxotrophs. The relative order of thrO (operator) mutations was established. We propose that an operator region is located between a promoter region and the structural genes. Mutations leading to the desensitization of the aspartokinase I-homoserine dehydrogenase I towards threonine were localized in two different regions of the thrA gene.

Aspartokinase Homoserine Dehydrogenase↗

PubMed1991

1H NMR and CD secondary structure analysis of cell adhesion promoting peptide F-9 from laminin.

A heparin binding, cell adhesion promoting domain, termed peptide F-9, from the B1 chain of human laminin, residues 641 to 660, i.e. RYVVLPRPVCFEKGMNYTVR, has been investigated by 1H NMR (500 MHz) spectroscopy and CD spectropolarimetry. While small linear peptides in water solution normally exist in a number of fluctuating conformational states, CD data analysis of peptide F9 indicates the existence of some preferred average structural populations consisting of about 30% beta-sheet, 22% beta-turn, and 6% alpha-helix. NMR structural analysis supports this observation and indicates specific sequences of preferred structural populations. Evidence for these is indicated by the presence of dNN nuclear Overhauser effect (NOE) populations and attenuated or absent d alpha N NOEs at short mixing times (0.1 s), 3J alpha N coupling constants of 5 and 10 Hz, and chemical shifts significantly removed from random coil positions. The NH2-terminal VVL sequence primarily exists in an extended chain conformation by virtue of large d alpha N NOEs and 9-10 Hz 3J alpha N coupling constants. Residues C10-N16 have turn-like or helix character with a run of dNN and d beta N NOEs and attenuated d alpha N NOEs. These midchain reversals include the lysine and asparagine residues proposed to be involved in heparin binding and N-glycosylation, respectively, to laminin peptide F-9.

Amino Acid Sequence↗

PubMed1990

Electrospray ionization mass spectrometric peptide mapping: a rapid, sensitive technique for protein structure analysis.

Electrospray ionization mass spectrometric peptide mapping is demonstrated to be a useful new technique for protein structure analysis. The procedure involves the digestion of the protein with trypsin and subsequent analysis of the total unfractionated digest by electrospray ionization mass spectrometry. The utility of the technique for investigating protein structure is illustrated by a peptide mapping analysis of human apolipoprotein AI (Mr = 28 kDa). The technique is rapid, sensitive, and requires no prior separation of the peptides. The discrimination effects observed in other mass spectrometric methods are less important in the present procedure.

Apolipoprotein A-I↗

PubMed1992

A group additivity model for analyzing absorption spectra of organic compounds: applications to partial structural analysis and molecular weight determinations of polymers, nucleotides, and peptides.

A new method for the analysis of organic structural groups from absorption spectra is described. The method is based on the integrated intensity of absorption. It requires only micrograms of analyzed compound, which may be fully recovered after analysis from its solution. The method can be used for different kinds of structural and/or kinetic studies. For example, the reaction kinetics and tautomeric equilibria can be easily studied by this method. The method can also be used for the determination of molecular weight and quantitative composition of polymers, nucleotides, and/or peptides. Hydrolysis or derivatization of the studied compounds is not necessary. On the basis of this method, automatic molecular weight, nucleotide, and peptide analyzers can be constructed.

Hydrogen-Ion Concentration↗

PubMed1985

Fine structural analysis of lipofuscin in various tissues of rats of different ages.

Comparative fine structural analysis of intracellular dense inclusions in various tissues of rats of different ages (2, 11 and 29.5 months old) was made. The majority of dense inclusions observed in 2-month-old rats were of the granular type. With advancing age, granular type inclusions gradually decreased in frequency, while compound type inclusions, such as granular-homogeneous and granular-lamellated type increased except for spleen. In the bronchial epithelia, granular-lamellated inclusions appeared at 2 months of age. On the other hand, in the heart, liver, spleen, and skeletal muscle, lamellated or granular-lamellated inclusions were not observed at any of the three age levels. Dense inclusions seemed to have various patterns of distribution, frequency of appearance, and source of organelles in each tissue and cell. They also did not show the maturity of pigment in the process of pigment formation. These findings suggested that these inclusions were not merely age-related granules, but seemed to be influenced by their relationship to the cell metabolism and other functions.

Aging↗