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At least 1,243 records · Page 69Linked to original sources
PubMed2000

Structure-function relationships in replication origins of the yeast Saccharomyces cerevisiae: higher-order structural organization of DNA in regions flanking the ARS consensus sequence.

In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae. Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS). We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence. The data show that this organization correlates with an active ACS. Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs. Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance. A model for the role of these individual structural components of the yeast replication origin is presented. We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function.

Base Sequence↗
PubMed2004

Comparison of outcomes between overlapping structure-based and non-overlapping structure-based optimization for simultaneous integrated boost IMRT for malignant gliomas.

BACKGROUND: Intensity-modulated radiotherapy (IMRT) can deliver different doses to two target volumes with high conformity. The purpose of the present study was to compare outcomes provided by two different optimization methods, overlapping structure-based and non-overlapping structure-based methods, for simultaneous integrated boost (SIB)-IMRT for malignant gliomas. METHODS: Treatment plans for three glioblastomas and one anaplastic astrocytoma were analyzed in the present study. The planning protocol was to deliver 70 Gy/28 fractions (fr) to the gross tumor volume (GTV) and 56 Gy/28 fr to the surrounding edema. Two different optimization methods were tested for optimizing dose distribution to the GTVand the surrounding edema. One method was the "including method", an overlapping structure-based (GTV and the clinical target volume [CTV]) optimization method. The other method was the "annulus method", a non-overlapping structure-based (GTV and the subtracted volume) optimization method. Dosimetric indexes derived from dose-volume histograms (DVHs) were used for the analysis. RESULTS: There was no significant difference between the two methods in the mean doses of the target volumes and the doses delivered to the 5% or 95% target volumes (D05 or D95). The mean dose to the brain by the including method was significantly higher than that delivered by the annulus method (P = 0.0001). The D05 of the brain showed no significant difference between the two methods. CONCLUSION: The two optimization methods provided comparable dose distributions within the target volumes and normal brain.

Brain Neoplasms↗
PubMed2005

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin.

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 A. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 A for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp561 could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.

Animals↗
PubMed1977

The structural organization of nuclear pre-mRNA. II. Very long double-stranded structures in nuclear pre-mRNA.

High molecular weight nuclear pre-messenger RNA (pre-mRNA or hnRNA) isolated from Ehrlich ascites carcinoma cells contains besides moderately long (100--200 base pairs) snap-back double-stranded structures, also longer double-stranded structure containing at least 300--800 base pairs. Their double-stranded nature was proved by Cs2SO4 gradient centrifugation. Very long double-stranded sequences are not able to snap-back after RNA melting. While the moderately long double-stranded RNA (dsRNA) is renatured at C0t1/2 approximately equal to 5-10(-4), the very long dsRNA shows a higher complexity (C0t1/2 approximately equal to 2-10(-2). They also hybridize to less reiterated class of DNA than moderately long dsRNA. Two classes of dsRNA are represented by different sequences as followed from cross-renaturation experiments. Very long dsRNA forms stable hybrids with 20% of total poly(A)+mRNA of cytoplasm. The properties of different classes of ds structures present in nuclear pre-mRNA are compared and their possible nature is discussed. The presence of very long dsRNA may reflect either the symmetric transcription of structural genes, or the transcription from those DNA sequences which are complementary to each other but located in different parts of the genome.

Animals↗
PubMed1979

Lanthanide ion probes of structure in biology. Environmentally sensitive fine structure in laser-induced terbium(III) luminescence.

The 488 nm line of the CW argon ion laser provides a convenient visible source for the direct excitation of the emissive 5D4 state of the Tb(III) ion. Room temperature emission spectra of Tb(III) in a variety of environments have been examined under relatively high resolution. The samples studied include structurally well-characterized crystalline solids, model chelate complexes in solution and Tb(III) bound to the enzyme thermolysin and the protein parvalbumin. The fine structure in the emissions is caused by ligand field splittings of both ground and excited state J manifolds. These spectra provide signatures sensitive to the immediate coordination environment of the Tb(III) ion. Solid state/solution state structural comparisons are made. The emission fine structure reveal differences between the EF side calcium-binding sites of parvalbumin and the calcium site 1 of thermolysin.

Calcium↗
PubMed1985

The structure of the voltage-sensitive sodium channel. Inferences derived from computer-aided analysis of the Electrophorus electricus channel primary structure.

A variety of computer-aided analyses was applied to the recently derived amino acid sequence of the Electrophorus electricus sodium channel protein in order to extract structural information such as hydrophobicity, periodicity, and secondary structure predictors. We propose a schematic model for the arrangement and folding of the polypeptide chain within the bilayer. The model consists of 4 homologous regions, each containing 8 membrane-spanning (probably alpha-helical) structures. Several of these structures are amphipathic with a repeat of 3.5 residues, 4 of which (one from each homologous region) are postulated to form a negatively charged channel lining. Gating currents are proposed to arise from voltage-dependent separation of multiple ion pairs buried within the hydrophobic, intramembranous protein interior.

Amino Acid Sequence↗
PubMed1990

Structural features of the McPC603 Fab fragment not defined in the X-ray structure.

The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system [(1989) Methods Enzymol. 178, 497-515]. We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure [J. Mol. Biol. 190, 593-604]. In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region. These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments. Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures.

Amino Acid Sequence↗
PubMed1992

Molecular modelling of the structures of endothelin antagonists. Identification of a possible structural determinant for ET-A receptor binding.

Computer-aided molecular modelling of the endothelin (ET-A) receptor antagonists, BQ-123 and BE-18257B, shows that they have very similar 3D structures. Parts of their 3D structures are also shown to match closely with that reported for residues 6-8 in endothelin-1. On the basis of these similarities (and with supporting evidence from literature data on endothelin structure-activity relationships) a structural determinant is proposed for ET-A receptor binding, and novel designs of peptide are suggested for providing more potent and selective ET-A receptor antagonists.

Amino Acid Sequence↗
PubMed1993

Heterocomplexes of tick-borne encephalitis structural and non-structural proteins.

The existence of the complex of structural and non-structural proteins in the tick-borne encephalitis (TBE) virus is shown. The complex was isolated from virus-containing cultural medium by immunoaffinity chromatography on monoclonal antibodies (MAbs). By enzyme immunoassay and immunoblotting with the use of appropriate MAbs it was demonstrated that this complex consists of structural (protein E), and non-structural (NS1) glycoproteins. Also, the trimer E-NS1-NS3 can be isolated. It is proposed that this trimer is the viral replicative complex.

Antibodies, Monoclonal↗
PubMed1994

Crystal structure of domain A of Thermus flavus 5S rRNA and the contribution of water molecules to its structure.

This is the first high resolution crystal structure of an RNA molecule made by solid phase chemical synthesis and representing a natural RNA. The structure of the domain A of Thermus flavus ribosomal 5S RNA is refined to R = 18% at 2.4 A including 159 solvent molecules. Most of the 2'-hydroxyl groups as well as the phosphate oxygens are involved either in specific hydrogen bonds in intermolecular contacts or to solvent molecules. The two U-G and G-U base-pairs are stabilized by H-bonds supplied via three water molecules to compensate for the lack of base-pair hydrogen bonds. The structure shows for the first time in detail the importance of highly ordered internal water in stabilizing an RNA structure.

Base Sequence↗
PubMed1989

ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure.

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.

Adenosine Triphosphate↗
PubMed1991

Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase.

The structure of lipoamide dehydrogenase from Azotobacter vinelandii has been refined by the molecular dynamics technique to an R-factor of 19.8% at 2.2 A resolution. In the final model, the root-mean-square deviation from ideality is 0.02 A for bond lengths and 3.2 degrees for bond angles. The asymmetric unit comprises two subunits, each consisting of 466 amino acid residues and the prosthetic group FAD, plus 512 solvent molecules. The last ten amino acid residues of both chains are not visible in the electron density distribution and they are probably disordered. The operation required to superimpose the two chains forming the dimer is a rotation of exactly 180 degrees with no translation component. The final model shows the two independently refined subunits to be very similar, except for six loops located at the surface of the molecule. The structure of each subunit of the enzyme consists of four domains with the catalytic centre located at the subunit interface. The reactive disulphide bridge, 48-53, is oxidized with S gamma of Cys53 located 3.5 A away from carbon C-4a of the isoalloxazine ring. The side-chain of His450' points its N epsilon 2 towards S gamma of Cys48 and is hydrogen bonded to the carboxylate of Glu455'. The FAD is bound in an extended conformation and the isoalloxazine ring is not completely planar with an angle between the pteridine and the benzene ring of 7.3 degrees in the first subunit and of 12.1 degrees in the second one. The overall folding of lipoamide dehydrogenase is very similar to that of glutathione reductase. However, a comparison of the two enzymes, which have only 26% sequence identity, reveals significant conformational differences. These concern the tertiary as well as the quaternary structure of the two molecules. In each subunit of lipoamide dehydrogenase the NAD-binding domain and the interface domain appear to be differently oriented with respect to the FAD-binding domain by 7.1 degrees and 7.8 degrees, respectively. The interface domain contains, in addition, major changes in tertiary structure. Furthermore, the two subunits forming the dimer appear to be shifted with respect to each other by more than 4 A, when the lipoamide dehydrogenase dimer is compared with that of glutathione reductase. In spite of all these changes at the tertiary and quaternary level the active sites of the enzymes, which occur at the dimer interface, appear to be remarkably similar.(ABSTRACT TRUNCATED AT 400 WORDS)

Azotobacter↗
PubMed1992

De novo design, synthesis and study of albebetin, a polypeptide with a predetermined three-dimensional structure. Probing the structure at the nanogram level.

The de novo polypeptide named albebetin was designed to form the tertiary fold that has not yet been observed in natural proteins. The design was based on the molecular theory of protein structures. The gene coding for this polypeptide was chemically synthesized. For the initial characterization of a protein structure, a new approach has been developed that uses only nanogram amounts of a polypeptide without its previous purification. This approach includes the biosynthesis of radiolabeled protein in a cell-free translation system with subsequent analysis of its compactness and structure by size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis. According to all tests used, albebetin has a compact stable structure.

Amino Acid Sequence↗
PubMed1992

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.

Anions↗
PubMed1992

Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.

Capsid↗
PubMed1992

Methylated cap structures in eukaryotic RNAs: structure, synthesis and functions.

There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated gamma phosphate (Mppp) cap structure. The TMG capped RNAs are capped with M7G during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs.

Animals↗
PubMed1992

Secondary structure formation precedes tertiary structure in the refolding of ribonuclease A.

The kinetics of refolding of ribonuclease A were monitored by circular dichroism (CD), tyrosine fluorescence and absorbance in the -40 to -10 degrees C range using a methanol cryosolvent. The native-like far-ultraviolet CD signal returned in the dead-time of the mixing, whereas the native absorbance and fluorescence signals returned in a multiphasic process at rates several orders of magnitude more slowly. Thus the secondary structure was formed much more rapidly than the tertiary structure. In addition, the absorbance signal showed evidence of an early intermediate in which one, or more, tyrosine residues was in a transiently more polar environment. A total of four kinetic phases were observed by absorbance in refolding, the slowest two of which had energies of activation consistent with proline isomerization. A refolding scheme involving initial hydrophobic collapse, concurrent with secondary structure formation, followed by much slower rearrangement to the native tertiary structure is proposed.

Circular Dichroism↗
PubMed1989

Evolution of large subunit rRNA structure. The 3' terminal domain contains elements of secondary structure specific to major phylogenetic groups.

Refined secondary structure models supported by phylogenetic evidence have been derived for the 3' terminal domain of large subunit rRNA (the region that exists as a separate 4.5 S molecular entity in chloroplast ribosomes) through a comparative analysis of all the pro- and eukaryotic sequences at present available. While several universally conserved features of secondary structure are found, a few diversified structural elements are also detected which are specific to one of the primary kingdoms, eubacteria, archaebacteria, or eukaryotes. Remarkably, some appear to be selectively preserved during the evolution of the primary kindgom, suggesting they represent functionally important structures. Thus, although the role of this 3' terminal domain in ribosomal function still remains unknown, its mode of sequence variation clearly points to a significant diversification of its function among the primary kindgoms.

Animals↗