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PubMed2006

Chromatographic and electrophoretic characterization of protein variants.

Almost all proteins are expressed in several variants, also known as isoforms. Individual protein variants differ by modifications of the individual amino acid side chains, or the N- or C-terminus. Typical modifications are glycosylation, phosphorylation, acetylation, methylation, deamidation or oxidation. It is of utmost interest to either get a quantitative picture of the variants of a particular protein or to separate the variants in order to be able to identify their molecular structure. Protein variants are present in native as well as in recombinant proteins. In the case of protein production it is interesting, how variants are generated during fermentation, purification processes, storage, and how present individual variants influence the biological activity. This review provides a comparison of chromatographic and electrophoretic separation methods to analyze and to prepare protein variants.

Chromatography↗
PubMed2002

NMR structural study of two-disulfide variant of hen lysozyme: 2SS[6-127, 30-115]--a disulfide intermediate with a partly unfolded structure.

The 15N-labeled recombinant hen lysozyme and two species of two-disulfide variants, denoted as 2SS[6-127, 30-115] and 2SS[64-80, 76-94], were studied by means of NMR spectroscopy. The former variant contains two disulfide bridges in the alpha-domain, while the latter has one disulfide bridge in the beta-domain and the other one at the interface between two domains. Resonance assignments were performed using 3D TOCSY-HSQC and NOESY-HSQC spectra. The 15N-1H-HSQC spectrum of 2SS[6-127, 30-115] was similar to that of recombinant lysozyme as a whole, although a number of cross-peaks disappeared. On the other hand, the HSQC spectrum of 2SS[64-80, 76-94] was characteristic of unfolded proteins. The structure of 2SS[6-127, 30-115] was thoroughly examined on the basis of NOE contacts determined by NMR spectroscopy. The structure of the alpha-domain was quite similar to that of authentic lysozyme, while the beta-domain was largely unstructured. However, NMR data clearly demonstrated that some residual structures exist in the beta-domain. The beta1 and beta2 strands were maintained stably as an antiparallel beta-sheet. In addition, the residues 55 and 56 were located in the vicinity of the end of the B-helix. Further, the C-helix was properly set with side-chains of I88, V92, K96, and V99 facing toward the hydrophobic core in the alpha-domain. These residual structures inherent in the amino acid sequence were evaluated concerning the folding process of lysozyme. Our experiments imply that the establishment of the backbone conformation ranging from residues 76-99 plays a key role in attaining the cooperativity between two domains required for the folding transition.

Alanine↗
PubMed2004

Inhibitor-resistant class A beta-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant.

A bacterial response to the clinical use of class A beta-lactamase inhibitors such as tazobactam and clavulanic acid is the expression of variant beta-lactamases with weaker binding affinities for these mechanism-based inhibitors. Some of these inhibitor-resistant variants contain a glycine mutation at Ser130, a conserved active site residue known to be adventitiously involved in the inhibition mechanism. The crystallographic structure of a complex of tazobactam with the Ser130Gly variant of the class A SHV-1 beta-lactamase has been determined to 1.8 A resolution. Two reaction intermediates are observed. The primary intermediate is an acyclic species bound to the reactive Ser70. It is poorly primed for catalytic hydrolysis because its ester carbonyl group is completely displaced from the enzyme's oxyanion hole. A smaller fraction of the enzyme contains a Ser70-bound aldehyde resulting from hydrolytic loss of the triazoyl-sulfinyl amino acid moiety from the primary species. This first structure of a class A beta-lactamase lacking Ser130, the side chain of which functions in beta-lactam binding and possibly in catalysis, gives crystallographic evidence that the acylation step of beta-lactam turnover can occur without Ser130. Unexpectedly, the crystal structure of the uncomplexed Ser130Gly enzyme, also determined to 1.8 A resolution, shows that a critical Glu166-activated water molecule is missing from the catalytic site. Comparison of this uncomplexed variant with the wild-type structure reveals that Ser130 is required for orienting the side chain of Ser70 and ensuring the hydrogen bonding of Ser70 to both Lys73 and the catalytic water molecule.

Alanine↗
PubMed2000

Structure and expression of the variant melanin-concentrating hormone genes: only PMCHL1 is transcribed in the developing human brain and encodes a putative protein.

PMCHL1 and PMCHL2 are two copies of the so-called variant melanin-concentrating hormone (MCH) gene that are located, respectively, on human chromosome 5p14 and 5q13 and that emerged recently during primate evolution. They correspond to a 5'-end truncated version of the MCH gene mapped on chromosome 12q23 and encoding a neuropeptide precursor. The gene organization and regulation of the expression of the variant MCH genes in the human brain are the central issues we investigated. First, the structure and fine chromosomal mapping of the 5p and 5q variant MCH genes were established. These revealed several point mutations and length variations of one CA/TA repeat which allow discrimination between each copy. Using a combination of RACE-PCR, RT-PCR, and sequencing analysis, we provided strong evidence for the expression of the PMCHL1 gene but not the PMCHL2 gene in the human fetal, newborn, and adult brains. Sense, potentially coding, RNAs, as well as noncoding antisense RNAs, were identified and displayed a region-specific expression in the human brain. Strikingly, sense unspliced RNAs of the PMCHL1 gene carried a novel open reading frame and may produce an NLS-containing protein of 8 kDa named VMCH-p8. These transcripts were translated in vitro and in transfected COS cells. Therefore, the PMCHL1 gene provides a unique example of the generation of a gene in the Hominoidae lineage which is specifically transcribed in the developing human brain and has the capacity to be translated into a putative novel protein.

Amino Acid Sequence↗
PubMed1996

Structure-function relationships of band 3 variants.

This review describes many of the naturally occurring band 3 variants that have been reported in the literature to date; from the common band 3 Memphis, to the rare band 3 HT. Both the molecular basis of these variants, and their effect on the structure and/or function of band 3, are described. The blood group antigens that have recently been assigned to band 3, such as Diego, Wright, Waldner, Redelberger and Warrior, are mentioned. Band 3 variants that affect the morphology of the red cell (e.g. acanthocytosis in band 3 HT and stomatocytic ovalocytosis in band 3 SAO) are described, as are many of the band 3 mutations that cause instability, either at the mRNA or protein level, and hence hereditary spherocytosis (HS). Band 3 variants that affect the binding pocket of the anion transport inhibitor, 4,4'-diisothiocyanato-2,2'-dihydrostilbene disulphonic acid (H2DIDS), (e.g. Diego and band 3 HT) and band 3 variants that affect the rate of anion transport (e.g. band 3 HT and band 3 in red cells that lack glycophorin A (GPA)) are reviewed in greater detail. The association between band 3 and GPA is discussed; both with respect to the Wright antigens and with regards the structure/function of band 3 in the absence of GPA.

Acanthocytes↗
PubMed2000

Laboratory investigation of hemoglobinopathies and thalassemias: review and update.

Structural hemoglobin (Hb) variants typically are based on a point mutation in a globin gene that produce a single amino acid substitution in a globin chain. Although most are of limited clinical significance, a few important subtypes have been identified with some frequency. Homozygous Hb C and Hb S (sickle cell disease) produce significant clinical manifestations, whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although heterozygotes for these variants are typically asymptomatic, diagnosis may be important for genetic counseling. Thalassemia, in contrast, results from quantitative reductions in globin chain synthesis. Those with diminished beta-globin chains are termed beta-thalassemias, whereas those with decreased alpha-chain production are called alpha-thalassemias. Severity of clinical manifestations in these disorders relates to the amount of globin chain produced and the stability of residual chains present in excess. The thalassemia minor syndromes are characterized clinically by mild anemia with persistent microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate, variably compensated hemolytic anemia that may present with clinical symptoms during a period of physiologic stress such as infection, pregnancy, or surgery. The thalassemia major syndromes produce severe, life-threatening anemia. alpha-Thalassemia major usually is incompatible with extrauterine life; beta-thalassemia major presents in infancy and requires life-long transfusion therapy and/or bone marrow transplantation for successful control of the disease. Double heterozygosity for certain structural variants and/or thalassemia syndromes may also lead to severe clinical disease. Several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation. The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders.

Chromatography, High Pressure Liquid↗
PubMed2004

Competitive adsorption of bacteriophage T4 lysozyme stability variants at hydrophilic glass surfaces.

The competitive adsorption behavior exhibited by the wild-type T4 lysozyme and two of its structural stability variants was studied by 125I radioisotope labeling. The mutant lysozymes were produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability, or with a cysteine residue, resulting in a protein with higher structural stability. Adsorption kinetics were recorded for binary protein mixtures in contact with a clean glass surface, in which one variant had been radiolabeled and the other had not. All pair permutations were tested. The kinetic data show that in instances in which exchange reactions between adsorbed protein and dissolved protein occur, they occur such that more stable variants are removed from the surface by less stable variants. The less stable proteins thus exhibited an advantage in competitive adsorption over the more stable proteins, in these tests.

Adsorption↗
PubMed2005

Chief cell hyperplasia with structural and nuclear atypia: a variant of fundic gland polyp.

A case of an unusual variant of fundic gland polyp (FGP) composed of chief cell hyperplasia with structural and nuclear atypia in an 87-year-old woman is presented. Gastrointestinal endoscopy revealed a sessile polyp in the cardia/ corpus transition zone and a polypoid lesion in the fundus. Histologically, the polyp in the cardia/corpus showed a typical appearance of FGP, while that in fundus demonstrated a tumorous lesion composed of irregular branched tubules with nuclear stratification. Despite the structural distortion and nuclear atypia, mitotic figures were absent and MIB-1 positive cells were less than 3%. Immunohistochemically, the cytoplasms of the tubules were negative for gastric mucin and Muc-5AC glycoprotein, but mostly positive for pepsinogen-I, indicating that the proliferated glands consisted mainly of chief cells, not mucous cells. Parietal cells were occasionally found in the glands. At the periphery of the lesion, microcysts composed of parietal cells, chief cells, and mucous cells had developed. Altogether, the polyp in the fundus was diagnosed as an unusual variant of FGP with chief cell hyperplasia. This FGP should be differentiated from tubular adenocarcinoma. Proliferation of chief cells with occasional parietal cells is critical for the differential diagnosis.

Adenocarcinoma↗
PubMed1976

[Morphological diagnosis of renal cell carcinoma. Histo-cytological parallels].

A detailed parallel histo-cytological investigation of 100 observations on renal cell carcinoma showed that these tumours had a various macro- and microscopic structure. For the substantiation of correct diagnosis material should be taken from several places of the tumour of different microscopic structure. In diagnosis all detected variants of the structure of the tumour and the degree of cellular anaplasia should be mentioned. A clear-cell variant in the pure form was revealed in 52% of cases, other one-type variants were very rare (7%). In tumours of a mixed structure (41%) there were observed various combinations of all the variants occurring in the order: clear-cell, granular-cell, sarcoma-like, glandular. Collation of Histological and cytological data showed that in the overwhelming majority of cases (97%) there was a complete coincidence in determination of the morphological type of renal cell carcinoma.

Adenocarcinoma↗
PubMed1987

Structural analysis of an HLA-B27 functional variant, B27d, detected in American blacks.

The structure of a new functional variant B27d has been established by comparative peptide mapping and radiochemical sequencing. This analysis completes the structural characterization of the six known histocompatibility leukocyte antigen (HLA)-B27 subtypes. The only detected amino acid change between the main HLA-B27.1 subtype and B27d is that of Tyr59 to His59. Position 59 has not been previously found to vary among class I HLA or H-2 antigens. Such substitution accounts for the reported isoelectric focusing pattern of this variant. HLA-B27d is the only B27 variant found to differ from other subtypes by a single amino acid replacement. The nature of the change is compatible with its origin by a point mutation from HLA-B27.1. Because B27d was found only in American blacks and in no other ethnic groups, it is suggested that this variant originated as a result of a mutation of the B27.1 gene that occurred within the black population.

Amino Acid Sequence↗
PubMed1999

Evaluation of charge derivatization of a proteolytic protein digest for improved mass spectrometric analysis: de novo sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry.

A simple mass spectrometric method to sequence a recombinant phosphoenolpyruvate carboxykinase of known structure and a novel variant of unknown structure isolated from Anaerobiospirillum succiniciproducens and Actinobacillus succinogenes 130Z, respectively, was evaluated. The proteolytic digests of the proteins were each chemically derivatized at the N-terminus by addition of a tris(trimethoxyphenyl)phosphoniumacetyl (TMPP(+)-Ac) group to produce peptides with a fixed positive charge. The derivatized digests were then partially separated by reversed-phase high-performance liquid chromatography. The fractions collected were subjected to matrix-assisted laser desorption/ionization post-source decay (MALDI/PSD) mass spectrometric analysis. The resulting spectra are sufficiently simple to allow the sequence to be read directly without extensive interpretation. This is in contrast to spectra of underivatized peptides obtained by MALDI/PSD or conventional tandem mass spectrometry, where full sequence interpretation can be challenging. Aided with a set of very simple established rules, it was shown that the sequence of TMPP(+)-Ac derivatives can be derived strictly from predictable fragment ion series. In most cases, this is sufficient to determine extensive, unambiguous, peptide sequences de novo. The partial sequence (35%) of the unknown phosphoenolpyruvate carboxykinase from Actinobacillus succinogenes 130Z was obtained entirely by the mass spectrometric method evaluated here, which provided the basis for evaluating homology and for the design of oligonucleotide probes for cloning the corresponding gene.

Amino Acid Sequence↗
PubMed1981

Hemoglobin connecticut (beta 21 (B3) Asp leads to Gly): a hemoglobin variant with low oxygen affinity.

Electrophoretic analysis of a hemolysate from a young man undergoing a routine physical examination revealed an abnormal hemoglobin with a mobility similar to Hb S on cellulose acetate (pH, 8.4). This new variant, designated Hb Connecticut, was found in three generations of a family of Polish descent. Several individuals possessing the variant exhibited mild anemia. Structural analysis of the abnormal beta-chain indicated that the amino acid substitution was at position 21 (B3), and involved the replacement of aspartic acid with glycine. Oxygen dissociation studies revealed low oxygen affinity. The alkaline Bohr effect and the degree of cooperativity were unchanged. Analysis of the crystal structure of the variant suggested that the low oxygen affinity was due to the possible disruption of salt bridges between aspartic acid 21(B3) and lysines 61(ES) and 65(E9), changes that could lead to steric interference in oxygen binding.

Adolescent↗
PubMed2000

Sc6MTe2 (M = Mn, Fe, Co, Ni): members of the flexible Zr6CoAl2-type family of compounds.

The compounds Sc6MTe2 (M = Mn, Fe, Co, Ni) have been prepared by high-temperature solid-state techniques and their structures determined to be hexagonal P62m (No. 189), Z = 1, a = 7.662(1) A, 7.6795(2) A, 7.6977(4) A, 7.7235(4) A and c = 3.9041(9) A, 3.8368(2) A, 3.7855(3) A, 3.7656(3) A for M = Mn, Fe, Co, and Ni, respectively. Crystal structures were refined for M = Fe and Ni, while M = Mn and Co were assigned as isostructural on the basis of powder diffraction data. The Sc6MTe2 compounds belong to a large family with the Zr6CoAl2-type structure, an ordered variant of the Fe2P structure. The structure contains confacial tricapped trigonal prisms of scandium centered alternately by the late transition metal or tellurium atoms. The Sc6MTe2 compounds are the electron-poorest examples of this structure type. Extended Hückel band calculations for M = Fe and Ni show that both compounds exhibit largely 1D metal-metal bonding and are predicted to be metallic.

Journal Article↗
PubMed2005

Three modulation patterns in four related [M(H2O)2(15-crown-5)](NO3)2 structures.

The structures of [M(H2O)2(15-crown-5)](NO3)2, M = Cu, Zn, Mg and Co, and 15-crown-5 = 1,4,7,10,13-pentaoxacyclopentadecane, have been redetermined at 294 and 90 K. The four structures, and a second form of the Cu structure, have been reported in the literature, but are all incorrect in some significant way. The structures, which all have at least two independent formula units (i.e. Z' > or = 2), are related; each water ligand is hydrogen-bonded to two nitrate anions, while each nitrate anion is hydrogen bonded to the water ligands of two cations. In the tetragonal Co structure the hydrogen-bond pattern is three-dimensional; in the monoclinic Cu, Zn and Mg structures the hydrogen-bond patterns are two-dimensional. In the isostructural Zn and Mg structures Z' = 3, while in the Cu structure Z' = 5. The Cu, Zn and Mg structures are modulated variants of a basic structure, which was reported for Cu but which probably does not exist. The conformations of the 11 independent cations are remarkably similar; they all have approximate twofold symmetry and so exist as conformational enantiomers. The most important modulation is imperfect enantiomeric alternation of the cations along the longest cell axis; the independent cations are related by very good pseudotranslation and pseudoinversion operations. The diffraction patterns for all four structures have classes of weak, even very weak, reflections.

Journal Article↗
PubMed1990

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids.

A number of eukaryotic surface glycoproteins, including the variant surface glycoproteins of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosyl-phosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated precursor glycolipid that can be transferred en bloc to the polypeptide. We have reported the purification and partial characterization of a candidate precursor glycolipid (P2) and of a compositionally similar glycolipid (P3) from T. brucei (Menon, A. K., Mayor, S., Ferguson, M. A. J., Duszenko, M., and Cross, G. A. M. (1988) J. Biol. Chem. 263, 1970-1977). The primary structure of the glycan portions of P2 and P3 have now been analyzed by a combination of selective chemical fragmentation and enzymatic glycan sequencing at the subnanomolar level. The glycans were generated by deamination, NaB3H4 reduction, and dephosphorylation of glycolipids purified from different trypanosome variants. Glycan fragments derived from biosynthetically labeled glycolipids were also analyzed. The cumulative data strongly suggest that P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The structural similarities suggest that GPI membrane anchors are derived from common precursor glycolipids that become variably modified during or after addition to newly synthesized proteins.

Animals↗
PubMed1978

Novel bacterial structures in human blood. II. Bacterial variants as etiologic agents in idiopathic hematuria.

Novel bacterial structures have been demonstrated in lysed blood filtrates placed in special culture media from patients with idiopathic hematuria. These structures converted rapidly to gram-positive coccal (streptococcal and staphylococcal-like), coccobacillary and filamentous, bacterial forms in vitro from 96 per cent of the patients studied. Blood cultured conventionally yielded negative findings. Although structures (dense bodies) were demonstrated in normal control blood specimens (albeit in lesser numbers) few converted to classical bacteria in vitro (7 per cent). Erythromycin therapy appeared to correlate with disappearance of hematuria and inability to revert rapidly the variant forms to classical bacteria in vitro. It is suggested that continual bombardment of the blood by bacteria entering from the mouth or other sites may lead to the development of variant bacterial parasitism. In an effort to survive the deleterious host effects the organisms may convert to persisting osmotically stable variant bacterial forms (dense bodies). Development of a disease state may be conditioned by some existing or developing abnormality in the host (immunologic, physiologic and/or biochemical). Furthermore, changes (genetic?) that might take place in the organisms per se during their transition to variant forms and adaptation to life in vivo may not allow certain host environments to adapt to these new forms, possibly leading to a pathogenetic role in renal diseases whose etiologies have long been enigmas.

Adolescent↗
PubMed1977

Novel bacterial structures in human blood. II. Bacterial variants as etiologic agents in idiopathic hematuria.

Novel bacterial structures have been demonstrated in lysed blood filtrates placed in special culture media from patients with idiopathic hematuria. These structures converted rapidly to gram-positive coccal (streptococcal and staphylococcal-like) coccobacillary and filamentous, bacterial forms in vitro from 96 per cent of the patients studied. Blood cultured conventionally yielded negative findings. Although structures (dense bodies) were demonstrated in normal control blood specimens (albeit in lesser numbers) few converted to classical bacteria in vitro (7 per cent). Erythromycin therapy appeared to correlate with disappearance of hematuria and inability to revert rapidly the variant froms to classical bacteria in vitro. It is suggested that continual bombardment of the blood by bacteria entering from the mouth or other sites may lead to the development of variant bacterial parasitism. In an effort to survive the deleterious host effects the organisms may convert to persisting osmotically stable variant bacterial forms (dense bodies). Development of a disease state may be conditioned by some existing or developing abnormality in the host (immunologic, physiologic and/or biochemical). Furthermore, changes (genetic?) that might take place in the organisms per se during their transition to variant forms and adaptation to life in vivo may not allow certain host environments to adapt to these new forms, possibly leading to a pathogenetic role in renal diseases whose etiologies have long been enigmas.

Adolescent↗
PubMed2000

Kinetic and structural characterization of urease active site variants.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.

Aspartic Acid↗