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PubMed1997

Detection of a genetic variant, lysine-->glutamic acid at position 372 of human serum albumin, by capillary electrophoresis and structural identification.

A genetic variant of human serum albumin (alloalbumin) is detected by capillary electrophoresis (CE). Two albumin peaks, which were in the ratio of approximately one, were clearly separated. One of the peaks had the same migration time as normal albumin (Alb A) and the other (Alb X) had a longer migration time. SDS-polyacrylamide gel electrophoresis of CNBr fragments (CB) of Alb X indicated that the amino acid substitution was localized in the CB5 fragment (residue 330-446) of the molecule, because of anomalous migration of CB5 in the gel. The CE mapping of the tryptic peptides from the variant CB5 revealed clearly the existence of a new peptide, and the lack of two normal peptides. The sequence analysis of the variant peptide collected by CE micropreparation showed that the N-terminus of the variant peptide corresponded to that of T49 in Alb A. The substitution site, lysine-->glutamic acid at the position 372, was revealed by sequence determination of the variant peptide purified by reversed-phase HPLC.

Amino Acid Sequence↗

PubMed2004

Unexpected diversity in the fine specificity of monoclonal antibodies that use the same V region gene to glucuronoxylomannan of Cryptococcus neoformans.

Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.

Antibodies, Fungal↗

PubMed2000

[Structural organization and evolution of t-complex in Mus genus].

Modern data on the structure and evolution of the t complex are discussed. The t complex is a series of inversions in the proximal region of murine chromosome 17; it contains a set of genes that determine its predominant transmission to the offspring of heterozygous males. Variants of structural organization of this genetic system (t haplotypes) have been found in wild populations of four species of genus Mus (M. domesticus, M. musculus, M. molossinus, and M. castaneus), but not in representatives of other, evolutionarily remote species of this genus. The so-called vertical, horizontal, and introgressive hypotheses are discussed of the origin and evolution of the t complex. Based on population genetic studies and molecular analysis a new hypothesis on the origin of the t-complex is put forward. This hypothesis is a synthesis between the vertical and horizontal models and assumes that all known t haplotypes had a common ancestral chromosome 17 carrying a proximal inversion.

Animals↗

PubMed2002

Structure of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor: evidence for strong structural coupling between receptor binding sites.

The structure of the ternary complex between the phage display- optimized, high-affinity Site 1 variant of human growth hormone (hGH) and two copies of the extracellular domain (ECD) of the hGH receptor (hGHR) has been determined at 2.6 A resolution. There are widespread and significant structural differences compared to the wild-type ternary hGH hGHR complex. The hGH variant (hGH(v)) contains 15 Site 1 mutations and binds>10(2) tighter to the hGHR ECD (hGH(R1)) at Site 1. It is biologically active and specific to hGHR. The hGH(v) Site 1 interface is somewhat smaller and 20% more hydrophobic compared to the wild-type (wt) counterpart. Of the ten hormone-receptor H-bonds in the site, only one is the same as in the wt complex. Additionally, several regions of hGH(v) structure move up to 9A in forming the interface. The contacts between the C-terminal domains of two receptor ECDs (hGH(R1)- hGH(R2)) are conserved; however, the large changes in Site 1 appear to cause global changes in the domains of hGH(R1) that affect the hGH(v)-hGH(R2) interface indirectly. This coupling is manifested by large changes in the conformation of groups participating in the Site 2 interaction and results in a structure for the site that is reorganized extensively. The hGH(v)- hGH(R2) interface contains seven H-bonds, only one of which is found in the wt complex. Several groups on hGH(v) and hGH(R2) undergo conformational changes of up to 8 A. Asp116 of hGH(v) plays a central role in the reorganization of Site 2 by forming two new H-bonds to the side-chains of Trp104(R2) and Trp169(R2), which are the key binding determinants of the receptor. The fact that a different binding solution is possible for Site 2, where there were no mutations or binding selection pressures, indicates that the structural elements found in these molecules possess an inherent functional plasticity that enables them to bind to a wide variety of binding surfaces.

Binding Sites↗

PubMed1988

The H1 and core histone subtypes: differential gene expression and varied primary structures.

The patterns of chromosomal proteins reflect in many cases the functional state of the respective cell type. The H1 histone group is particularly important in this respect, since these histones are involved in the higher order chromatin organization above the level of chains of nucleosomes. In mammals, the H1 histone family comprises at least five main subtypes (H1a-H1e), a testicular variant (H1t) and, thirdly, a subtype H1(0), which is found only in terminally differentiated cells. The H1(0) variant is structurally related to the avian red blood cell specific histone H5, which was the basis for our recent isolation of the human H1(0) gene. Changes of H1 histone patterns may be crucial events in modulating local chromatin arrangements, since the formation of higher order chromatin structures depends on a cooperative interaction of the H1 histones. Variations in their patterns can be studied in vivo during several developmental processes (such as spermatogenesis, erythropoiesis, maturation of several cell types) or in vitro in several tumor cell lines upon treatment with several inducers or upon inhibition of cell division. The differential regulation of the individual H1 subtypes is reflected in the gene and mRNA structures coding for the respective proteins. The cell cycle regulated histones are mostly encoded by non-polyadenylated mRNAs, whereas H5 as well as H1(0) mRNA shows a poly(A) tail at its 3' end. In conclusion, gene activity may not only be controlled at the level of RNA polymerases and their regulatory transcription factors. The varied patterns of chromosomal proteins at different stages during development and differentiation suggest that the local or overall organization of chromatin plays an additional role in these regulatory programs. Hence, the analysis of variations in patterns of chromosomal proteins is an integral part of the investigation of gene regulation mechanisms.

Amino Acid Sequence↗

PubMed2006

Two crystal structures of Trichoderma reesei hydrophobin HFBI--the structure of a protein amphiphile with and without detergent interaction.

Hydrophobins are small fungal proteins that are highly surface active and possess a unique ability to form amphiphilic membranes through spontaneous self-assembly. The first crystal structure of a hydrophobin, Trichoderma reesei HFBII, revealed the structural basis for the function of this amphiphilic protein--a patch consisting of hydrophobic side chains on the protein surface. Here, the crystal structures of a native and a variant T. reesei hydrophobin HFBI are presented, revealing the same overall structure and functional hydrophobic patch as in the HFBII structure. However, some structural flexibility was found in the native HFBI structure: The asymmetric unit contained four molecules, and, in two of these, an area of seven residues was displaced as compared to the two other HFBI molecules and the previously determined HFBII structure. This structural change is most probably induced by multimer formation. Both the native and the N-Cys-variant of HFBI were crystallized in the presence of detergents, but an association between the protein and a detergent was only detected in the variant structure. There, the molecules were arranged into an extraordinary detergent-associated octamer and the solvent content of the crystals was 75%. This study highlights the conservation of the fold of class II hydrophobins in spite of the low sequence identity and supports our previous suggestion that concealment of the hydrophobic surface areas of the protein is the driving force in the formation of multimers and monolayers in the self-assembly process.

Amino Acid Sequence↗

PubMed1998

Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site.

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.

Base Sequence↗

PubMed1985

Structure of a duck H3 variant histone gene: a H3 subtype with four cysteine residues.

A duck recombinant DNA phage library was screened for H3 histone genes, and the sequence of a variant H3 gene, which appears not to be part of a histone gene cluster, has been determined. As derived from the nucleotide sequence, this gene codes for a 135-amino acid (aa) protein (as any other H3) and shows 10 aa substitutions compared with most published H3 structures. Six of these aa changes are based on one nucleotide (nt) substitutions in arginine codons. This results in three new histidines and, in addition to the highly conserved cysteine at position 110, three more cysteines are found in this H3 histone subtype.

Amino Acid Sequence↗

PubMed1998

Structure and stability of the P93G variant of ribonuclease A.

The peptide bonds preceding Pro 93 and Pro 114 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 A. This structure is essentially identical to that of the wild-type protein, except for the 91-94 beta-turn containing the substitution. In the wild-type protein, the beta-turn is of type VIa. In the P93G variant, this turn is of type II with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of delta deltaGm which reports on the stability lost in the variants, is 1.5-fold greater for the P114G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type II turn, which has a preference for a glycine residue in its i + 2 position.

Amino Acid Substitution↗

PubMedDate not supplied

Prominent crista terminalis mimicking a right atrial mixoma: cardiac magnetic resonance aspects.

A 68-year-old woman came to our observation with a clinical history of isolated systolic hypertension poorly controlled by the combination of ramipril 5 mg and hydrochlorothiazide 12.5 mg o.d. The ECG showed sinus rhythm with heart rate of 68 beats per minute and signs of left ventricular hypertrophy without strain. Further investigation included an echocardiogram that showed normal left and right cavities and normal cardiac valves. At the level of the posterior wall of the right atrial (RA) an apparent smooth, bean-like tumor, having a thin pedicle, was identified as a RA mixoma. Cardiac MRI was requested and showed in two sequential slices a muscular ridge, identified as a prominent crista terminalis. Some para-physiological structures sited in the RA may have the appearance of tumors, as crista terminalis, Eustachian valve extending into the RA chambers and Chiari network. The multiplain projections of MRI allow the cardiologist to identify the presence of intracardiac masses and to make a differential diagnosis between neoplasms and variant anatomic structures.

Aged↗

PubMed1986

A unique nucleoprotein structure associated with the Drosophila melanogaster 18-28 S rDNA nontranscribed spacer.

We have detected unique nucleoprotein particles specific for the 18-28 S rDNA nontranscribed spacer of Drosophila melanogaster. The particles migrate between di- and trinucleosomes on nucleoprotein gels, and are between mono- and dinucleosomal in DNA length. These migration properties suggest that the nontranscribed spacer particles could have a protein component larger than a histone core. The variant nucleoprotein structures map primarily within the nontranscribed spacer 235 base pair internal subrepeat, which is AT-rich and possesses a 50 base pair sequence homologous to the RNA polymerase I binding site.

Animals↗

PubMed1998

Echogenic structures in the left atrioventricular groove: diagnostic pitfalls.

A variety of echogenic structures that can be detected by echocardiography in the left atrioventricular groove are best seen in the parasternal long-axis view and can be missed if not carefully looked for. Normal anatomic structures or variants can be seen, for example, dilated coronary sinus, persistent left superior vena cava, or lipomatous tissue. Degenerative or inflammatory processes of the mitral annulus (calcification of the mitral annulus, mitral annular abscess) and aneurysms of the left circumflex coronary artery or of the descending aorta can depict pathologic structures. Hiatal hernia and tumors can be found as well.

Aortic Aneurysm, Thoracic↗

PubMed1997

NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64-->V + G mutant.

The 64 amino acid hirudin-like peptide HM2 (Hirudinaria manillensis) is one of the agents known to specifically block the blood-clotting enzyme thrombin, and therefore is used as a potential pharmacological tool for the treatment of arterial and venous thrombosis. This peptide and its derivatives provide a new set of probes for studies aimed at elucidating the structural basis of the inhibition of alpha-thrombin. We used 581, 699, and 492 nmr-derived constraints respectively in a protocol employing simulated annealing, followed by restrained molecular dynamics and restrained energy minimization to derive the three-dimensional structures of HM2 and its mutants the HM2 (V + G) and the HM2 (1-47). HM2 consists of a well-defined core region of two double-stranded beta-sheet and a disordered C-terminus. These features are shared by other members of the hirudin family. The same type of folding has also been observed for recombinant hirudins whose structure has been determined in solution by nmr spectroscopy and in the structure of the complex hirudin-thrombin determined by x-ray diffraction. Molecular dynamics (MD) simulation methods were applied in the study of the structural and dynamic fluctuation properties of the hirudin derivatives solvated by 1625 and 1276 water molecules with periodic boundary conditions for HM2 and HM2 (1-47), respectively. Trajectories of 100 and 50 ps for the two unconstrained systems were generated at constant temperature and pressure. Analysis of the MD simulation shows that the structure of the peptide core is fairly rigid and stable in itself while the conformation of the C-terminal tail, which is involved in the inhibitory mechanism of thrombin, fluctuates and appears as a disordered region.

Amino Acid Sequence↗

PubMed1995

Limited and defined truncation at the C terminus enhances receptor binding and degranulation activity of the neutrophil-activating peptide 2 (NAP-2). Comparison of native and recombinant NAP-2 variants.

We have previously described a C-terminally truncated variant of the chemokine neutrophil-activating peptide 2 (NAP-2) that exhibited higher neutrophil-stimulating capacity than the full-size polypeptide. To investigate the impact of the NAP-2 C terminus on biological activity and receptor binding, we have now purified the novel molecule to homogeneity. Furthermore, we have cloned, expressed in Escherichia coli, and purified full-size recombinant NAP-2 (rNAP-2-(1-70)) and a series of C-terminally deleted variants (rNAP-2-(1-69) to rNAP-2-(1-64)). Biochemical and immunochemical analyses revealed that the natural NAP-2 variant was structurally identical to the rNAP-2-(1-66) isoform. As compared with their respective native and recombinant full-size counterparts, both molecules exhibited approximately 3-4-fold enhanced potency in the induction of neutrophil degranulation as well as 3-fold enhanced binding affinity for specific receptors on these cells. All other variants were considerably less active. The natural occurrence of a NAP-2 variant truncated by exactly four residues at the C terminus suggests that limited and defined proteolysis at this site plays a role in the regulation of the biological function of the chemokine.

Amino Acid Sequence↗

PubMed2001

Structural characterization of two CD1A allelic variants.

CD1 molecules are specialized in presenting lipidic antigens to T lymphocytes. They are structurally and evolutionary related to MHC molecules and show very limited polymorphism. We have previously described and partially characterized a new human CD1A allele differing from the wild type CD1A by a substitution of Cysteine by Tryptophan at position 52 in the alpha1 domain of the CD1A molecule. The frequency of this allele varies from 10% in individuals of Caucasian origin to 56% in Chinese people. The aim of the present work was to structurally characterize this CD1A allele. To do this we have cloned and sequenced the full-length cDNA encoding the new CD1A allele. The cDNA sequence of this allele encodes a protein differing the wild type in two amino acids at positions 14 (Threonine versus Isoleucine) and 52 (Cysteine versus Tryptophan). The cDNAs encoding both wild type and mutant CD1A were cloned in the expression vector pSRalphaNeo and transfected into C1R and L721.221 cells. Cell surface expression of the protein products in transfected cell lines were analyzed by flow cytometry and immunoprecipitation using CD1a-specific monoclonal antibodies. Our results indicate that both allelic products are efficiently expressed on the cell surface.

Alleles↗

PubMed1996

Cloning of novel kinectin splice variants with alternative C-termini: structure, distribution and evolution of mouse kinectin.

The analysis of cDNA clones encoding novel variant forms of mouse kinectin, an endoplasmic reticulum (ER)-bound receptor for the motor protein kinesin, is reported. Kinesin and cytoplasmic dynein are involved in mediating the anterograde and retrograde movements of intracellular vesicles along the microtubule network. The amino acid sequence deduced from kinectin cDNA isolated from mouse spleen cell and testis libraries revealed a long signal peptide or transmembrane sequence, and a 328 amino acid residue globular N-terminal domain adjacent to a much larger 858-999-residue C-terminal coiled-coil rod domain. The C-terminal domain was composed of 18 coiled-coil regions formed from multiple contiguous heptad repeats which undergo alternative splicing as evidenced by the presence of at least five small (23-33 amino acid residue) insertion sequences scattered throughout. The inserts are present in any one of a number of combinations, generating an array of novel kinectin variants. Insert 5 contains a termination codon, producing a C-terminus that is highly homologous to that of human kinectin. Three out of five mouse kinectin clones lack insert 5, generating a novel eleven amino acid C-terminus encoded by sequence that extends past the insertion site. The existence of alternative C-termini may have functional relevance given that the C-termini are exposed for interaction with kinesin, whereas the globular N-terminus is embedded in the ER membrane. Alternative C-termini represent candidate modifications that could determine specificity of binding to kinesin or cytoplasmic dynein, and the switching of directionality of movement. The cDNA hybridized to 4.5 kb transcripts expressed in all mouse cell lines and tissues examined, which provides the first indication that the kinectins are very widely distributed. Mouse kinectin is 42% similar over a 203 amino acid region to the chicken extracellular cardiac morphogen ES/130, whose canine homologue containing an inserted sequence of 10 amino acids repeated 54 times in tandem, is a ribosome receptor expressed on the ER. Mouse kinectin shares 64 and 83% identity, respectively, with its M(r) 160000 chicken and human kinectin homologues. There is a two-fold molar excess of kinectin over kinesin in unextracted vesicles, suggesting that kinectin might be a dimer. The electrostatic properties of the coiled-coil region of mouse kinectin, together with the relative frequencies of residues in particular positions within the heptad repeats support this notion.

Alternative Splicing↗

PubMed2005

Conformational heterogeneity at position U37 of an all-RNA hairpin ribozyme with implications for metal binding and the catalytic structure of the S-turn.

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.

Crystallization↗

PubMed1995

Solution structure of a human cystatin A variant, cystatin A2-98 M65L, by NMR spectroscopy. A possible role of the interactions between the N- and C-termini to maintain the inhibitory active form of cystatin A.

The solution structure of a human cystatin A variant, cystatin A2-98 M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by sD/3D heteronuclear double- and triple-resonance NMR spectroscopy. The structure is based on a total of 1343 experimental restraints, comprising 1139 distance, 154 phi and chi 1 torsion angle restraints, and 50 distance constraints for 25 backbone hydrogen bonds. A total of 15 structures was calculated using the YASAP protocol with X-PLOR, and the atomic rms distribution about the mean coordinate positions for residues 8-93 was 0.55 +/- 0.10 A for the backbone atoms and 1.05 +/- 0.11 A for all heavy atoms. The structure consists of five antiparallel beta-sheets and two short alpha-helices. Comparison with the X-ray structure of cystatin B in the papain complex shows that the conformation of the first binding loop is quite similar to that of cystatin A, with an rms deviation of 0.78 A for the backbone atoms in the 43-53 region (cystatin A numbering). The second binding loop, however, is significantly different in the two structures, with an rms deviation greater than 2 A. There are some other significant differences, especially for the N-terminal and alpha-helix regions. The overall structure of cystatin A is also compared with the recently reported NMR structure of the wild-type cystatin A (stefin A) at pH 5.5 (Martin et al., 1995) and reveals the following features. that differ in our structure from the previous one: (1) the N-terminal segment, which was unstructured in the previous report, folds over in close vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short alpha-helices linked by a type II reverse turn were found, instead of the continuous single alpha-helix with a slight kink shown in the previous structure; (3) the second binding loop, which was not well converged in the previous study at pH 5.5, is determined very well in our structure. The effect of the N-terminal truncation on the cystatin A structure was examined by comparing the 1H-15N HSQC spectrum of cystatin A2-98 with that of the cystatin A5-98 variant, which lacks the anti-papain activity, revealing significant chemical shift differences in the residual N-terminal segment and the first binding loop, together with small shifts in the other parts.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗