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PubMed2006

Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31.

The crystal structure of alpha-glucosidase MalA from Sulfolobus solfataricus has been determined at 2.5Angstrom resolution. It provides a structural model for enzymes representing the major specificity in glycoside hydrolase family 31 (GH31), including alpha-glucosidases from higher organisms, involved in glycogen degradation and glycoprotein processing. The structure of MalA shows clear differences from the only other structure known from GH31, alpha-xylosidase YicI. MalA and YicI share only 23% sequence identity. Although the two enzymes display a similar domain structure and both form hexamers, their structures differ significantly in quaternary organization: MalA is a dimer of trimers, YicI a trimer of dimers. MalA and YicI also differ in their substrate specificities, as shown by kinetic measurements on model chromogenic substrates. In addition, MalA has a clear preference for maltose (Glc-alpha1,4-Glc), whereas YicI prefers isoprimeverose (Xyl-alpha1,6-Glc). The structural origin of this difference occurs in the -1 subsite where MalA residues Asp251 and Trp284 could interact with OH6 of the substrate. The structure of MalA in complex with beta-octyl-glucopyranoside has been determined. It reveals Arg400, Asp87, Trp284, Met321 and Phe327 as invariant residues forming the +1 subsite in the GH31 alpha-glucosidases. Structural comparisons with other GH families suggest that the GH31 enzymes belong to clan GH-D.

Amino Acid Sequence↗

PubMed2004

Platelet factor 4 and interleukin-8 CXC chemokine heterodimer formation modulates function at the quaternary structural level.

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

Animals↗

PubMed2005

Single amino acid mutations alter the distribution of human porphobilinogen synthase quaternary structure isoforms (morpheeins).

Porphobilinogen synthase (PBGS) is an obligate oligomer that can exist in functionally distinct quaternary states of different stoichiometries, which are called morpheeins. The morpheein concept describes an ensemble of quaternary structure isoforms wherein different structures of the monomer dictate different multiplicities of the oligomer (Jaffe, E. K. (2005) Trends Biochem. Sci. 30, 490-497). Human PBGS assembles into long-lived morpheeins and has been shown to be capable of forming either a high activity octamer or a low activity hexamer (Breinig, S., Kervinen, J., Stith, L., Wasson, A. S., Fairman, R., Wlodawer, A., Zdanov, A., and Jaffe, E. K. (2003) Nat. Struct. Biol. 10, 757-763). All PBGS monomers contain an alphabeta-barrel domain and an N-terminal arm domain. The N-terminal arm structure varies among PBGS morpheeins, and the spatial relationship between the arm and the barrel dictates the different quaternary assemblies. We have analyzed the structures of human PBGS morpheeins for key interactions that would be predicted to affect the oligomeric assembly. Examples of individual mutations that shift assembly of human PBGS away from the native octamer are R240A and W19A. The alternate morpheeins of human PBGS variants R240A and W19A are chromatographically separable from each other and kinetically distinct; their structure and dynamics have been characterized by native gel electrophoresis, dynamic light scattering, and analytical ultracentrifugation. R240A assembles into a metastable hexamer, which can undergo a reversible conversion to the octamer in the presence of substrate. The metastable nature of the R240A hexamer supports the hypothesis that octameric and hexameric morpheeins of PBGS are very close in energy. W19A assembles into a mixture of dimers, which appear to be stable.

Amino Acid Substitution↗

PubMed2001

The X-ray structure determination of bovine carbonmonoxy hemoglobin at 2.1 A resoultion and its relationship to the quaternary structures of other hemoglobin crystal froms.

Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.

Animals↗

PubMed2003

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase.

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.

Allosteric Site↗

PubMed2001

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

Amino Acid Sequence↗

PubMed2004

Light-induced structural changes of LOV domain-containing polypeptides from Arabidopsis phototropin 1 and 2 studied by small-angle X-ray scattering.

Phototropin is a blue-light receptor of plants and comprises two light-receptive domains, LOV1 and LOV2, Ser/Thr kinase domain and one linker region connecting the LOV2 and the kinase domains. The LOV2 domain is thought to regulate predominantly the light-dependent autophosphorylation of the kinase domain, leading to cellular signaling cascades. In this study, we constructed recombinant LOV1, LOV2, and LOV2-linker polypeptides from phototropin 1 and phototropin 2 of Arabidopsis thaliana and studied their quaternary structures and light-dependent conformational changes by small-angle X-ray scattering. The molecular weights of the polypeptides determined from scattering intensities demonstrated the dimeric associations of LOV1 polypeptides of both isoforms. In contrast, while LOV2 and LOV2-linker polypeptides of phototropin 1 were homodimers, corresponding polypeptides of phototropin 2 existed as monomeric forms. Under blue-light irradiation, the LOV2-linker polypeptide of phototropin 1 displayed small but definite changes of the scattering profile. Through simulation of low-resolution molecular structures, the changes were likely explained as structural changes of the linker region and/or a movement of the region relative to the LOV2 domain. Light-induced profile changes were not observed in the Cys(512)Ala mutated LOV2-linker polypeptide of phototropin 1 losing the phototransformation capability. Thus, it was indicated that the photoreaction in the LOV2 domain probably caused the structural changes in the LOV2-linker polypeptide of phototropin 1. On the basis of the results, the interdomain interactions in phototropin are discussed.

Alanine↗

PubMed2001

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure.

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.

Amino Acid Sequence↗

PubMed2001

The position of the alpha and beta subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes.

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common alpha subunit and a hormone-specific beta subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the beta and alpha subunits are linked (NH(2)-CGbeta-alpha; CGbetaalpha orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of alpha was fused to the CGbeta amino terminus (NH(2)-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected Chinese hamster ovary cells was less than that seen for CGbetaalpha. The alphaCGbeta tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in alphaCGbeta, it was co-transfected with a monomeric alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating that CG dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.

Animals↗

PubMed2002

Importance of potential interhelical salt-bridges involving interior residues for coiled-coil stability and quaternary structure.

Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. Polar interactions involving residues at the interior a and d positions are important for determining the quaternary structure of coiled coils. In the model heterodimeric coiled-coil Acid-a1-Base-a1, a buried a-d' Asn-Asn interaction is sufficient to specify both a dimeric structure and an antiparallel relative helix orientation. Although the equivalent a-a' interaction is found in parallel coiled coils, there is no example of an a-d' Asn-Asn interaction in structurally characterized, naturally occurring antiparallel coiled coils. Instead, interior charged residues form interhelical salt-bridges with residues at the adjacent e or g positions. Using a model coiled-coil heterodimer, we have explored the role of a potential interhelical interaction between an Arg at an interior d position and a Glu at the adjacent g' position. Our results demonstrate that this potentially attractive interhelical Coulombic interaction has little or no influence on helix orientation. Instead, we show that burying a single Arg residue at an interior position is sufficient to specify a dimeric state at a significantly lower thermodynamic cost than burial of two interacting Asn residues.

Amino Acid Sequence↗

PubMed2003

Quaternary structure and catalytic activity of the Escherichia coli ribonuclease E amino-terminal catalytic domain.

RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria. Most endoribonucleases have been shown to act distributively; however, Feng et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746-14751] have recently found that RNase E acts via a scanning mechanism. A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E. coli RNase E forms a homotetramer. Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue. The tetrameric assembly of the N-terminal domain of RNase E is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D(2) dihedral symmetry. Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer. This shape, together with limited proteolysis data, suggests that the S1-RNA binding domains of RNase E lie on the periphery of the tetramer. These observations have implications for the structure and function of the RNase E/RNase G ribonuclease family and for the assembly of the E. coli RNA degradosome, in which RNase E is the central component.

Amino Acid Sequence↗

PubMed2002

SDS-resistant active and thermostable dimers are obtained from the dissociation of homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus in SDS. Stabilizing role of the A-C intermonomeric interface.

beta-Glycosidases are fundamental, widely conserved enzymes. Those from hyperthermophiles exhibit unusual stabilities toward various perturbants. Previous work with homotetrameric beta-glycosidase from hyperthermophilic Sulfolobus solfataricus (M(r) 226,760) has shown that addition of 0.05-0.1% SDS was associated with minimal secondary structure perturbations and increased activity. This work addresses the effects of SDS on beta-glycosidase quaternary structure. In 0.1-1% SDS, the enzyme was dimeric, as determined by Ferguson analysis of transverse-gradient polyacrylamide gels. The catalytic activity of the beta-glycosidase dimer in SDS was determined by in-gel assay. A minor decrease of thermal stability in SDS was observed after exposure to temperatures up to 80 degrees C for 1 h. An analysis of beta-glycosidase crystal structure showed different changes in solvent-accessible surface area on going from the tetramer to the two possible dimers (A-C and A-D). Energy minimization and molecular dynamics calculations showed that the A-C dimer, exhibiting the lowest exposed surface area, was more stabilized by a network of polar interactions. The charge distribution around the A-C interface was characterized by a local short range anisotropy, resulting in an unfavorable interaction with SDS. This paper provides a detailed description of an SDS-resistant inter-monomeric interface, which may help understand similar interfaces involved in important biological processes.

Binding Sites↗

PubMed2005

Phospholamban pentamer quaternary conformation determined by in-gel fluorescence anisotropy.

We measured in-gel fluorescence anisotropy of phospholamban (PLB) labeled with the biarsenical fluorophore FlAsH at three different sites on the cytoplasmic domain. The 6 kDa monomer bands of FlAsH-tetracysPLB showed high anisotropy (r = 0.29), reflecting null homotransfer and low mobility (S = 0.85) on the nanosecond time scale of the FlAsH fluorescence lifetime. 30 kDa bands (pentameric PLB) within the same lanes exhibited low anisotropy, suggesting intrapentameric fluorescence energy homotransfer between PLB subunits. FlAsH labels positioned at residue -6, 5, or 23 showed a graduated pattern of fluorescence depolarization corresponding to resonance energy transfer radii of 46 +/-2, 38 +/- 4, and <25 A, respectively. Pentamer anisotropy increased with heating or fluorescence photobleaching toward a maximum value similar to that determined for monomeric PLB. Fluorescence resonance energy heterotransfer was also observed in vitro and in vivo within PLB pentamers colabeled with FlAsH and the biarsenical fluorophore ReAsH. In vitro heterotransfer efficiencies were graduated by labeling position, in harmony with homotransfer results. The calculated transfer radii compare favorably to distances predicted by a computer molecular model of the phospholamban pentamer constructed from NMR solution structures. The data support a helical pinwheel model for the PLB pentamer, in which the cytoplasmic domains bend sharply outward from the central bundle of helices.

Arsenicals↗

PubMed2006

Flexible docking of an amyloid-forming peptide from beta(2)-microglobulin.

Using an all-atom, molecular dynamics-based, flexible docking method, the tertiary and quaternary structures of protofilaments of the "K3" fragment from beta(2)-microglobulin (residues Ser20-Lys41) were predicted at low pH in a continuous mixture of water and 2,2,2-trifluoroethanol (TFE). Tetramers with energies very close to the global minimum were produced with C(alpha) root-mean square deviation values under 4A over 88 residues compared to a recently solved SSNMR structure. The most accurate model distinguishes itself from other low-energy solutions in that it shows high structural similarity to another known fold, the parallel beta-helix, in agreement with models proposed previously by several other groups. The method achieves efficiency without loss of generality or atomic detail by enforcing local symmetry on the individual peptides, rewarding intermolecular contacts, and iteratively building up the protofilaments by successively doubling the number of chains. Solvent effects were included in the model by treating the dielectric constant and surface tension as functions of the TFE concentration. In order to understand the physical basis for the stabilizing effects of TFE, the TFE concentration was varied from 0% to 50% (v/v) and a peak in stability was observed at 16%, where the polar and hydrophobic terms cancel out and close to the experimentally determined value of 20%.

Amyloid↗

PubMed2000

Conserved structural features in class I major fimbrial subunits (Pilin) in gram-negative bacteria. Molecular basis of classification in seven subfamilies and identification of intrasubfamily sequence signature motifs which might Be implicated in quaternary structure.

Type 1 and P-pili are prototype members of Class I fimbriae produced by Gram-negative bacteria. Despite common structural characteristics, the low level of amino acid sequence conservation among the Class I major fimbrial subunits (pilins) indicates considerable evolutionary distance between members of this superfamily. We highlight here structural relatedness between Class I pilins from their two-dimensional sequence analysis using hydrophobic cluster analysis (HCA) and secondary structure predictions (PHD program). We present evidence that all members of the Class I pilin family have clear structural relatedness and suggest that classification based on phylogenetic analysis of Class I pilins into seven subfamilies correlates with differences in structural properties of the amino acid sequences. Using a sensitive alignment process (HCA), we identified 29 residues in topohydrophobic positions which probably play a prominent role in folding. The most striking aspects that distinguish the different pilin subfamilies are (i) large variation in the length of the loops connecting the structurally conserved regions and (ii) intrasubfamily sequence signature motifs located on regions predicted to be in the beta-conformation. We suggest that these "intrasubfamily sequence signature motifs" are part of interactive surfaces which participate in subunit-subunit interactions. These motifs prove highly useful in characterizing and classifying new Class I fimbriae that have not yet been described and whose sequence diverges appreciably from those of characterized groups. (After the submission of our manuscript, the experimental structure of Class I pilus subunits was published. In light of these actual pilin structures, a comparison has been made between the predicted results and the crystal structure in the Note Added in Proof.)

Amino Acid Motifs↗

PubMed2003

Quaternary structure of Azospirillum brasilense NADPH-dependent glutamate synthase in solution as revealed by synchrotron radiation x-ray scattering.

Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.

Animals↗

PubMed2005

Characterization of a large subunit catalase truncated by proteolytic cleavage.

The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to a structure similar to small subunit catalases. Mass spectrometry analysis indicates that there is some heterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminal residues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structure refinement at 2.8 A reveals that the tertiary and quaternary structure of the native enzyme is retained with only very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8 times faster turnover rate and enhanced sensitivity to high concentrations of H(2)O(2), consistent with easier access of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition, particularly by reagents such as aminotriazole and azide which are larger than substrate H(2)O(2). The main channel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortened and its entrance widened by removal of the C-terminal domain, providing an explanation for easier access to the active site. Opening of the entrance to the lateral channel also opens the putative NADPH binding site, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compound I species of both native and truncated HPII are reduced back to the resting state with compound II being evident in the absorbance spectrum only of the heme b-containing H392A variant.

Binding Sites↗

PubMed2001

Heat-induced quaternary transitions in hetero- and homo-polymers of alpha-crystallin.

PURPOSE: To compare the effects of heat incubation on the structure and function of native alpha-crystallin, urea denatured/renatured alpha-crystallin, and alphaA and alphaB-crystallin homo-polymers purified from bovine lenses. METHODS: Each of the alpha-crystallin samples were incubated for 1 h at temperatures ranging from 35 degrees C to 70 degrees C. After heat incubation structural perturbations in each of the samples were studied using non-denaturing gel electrophoresis, transmission electron microscopy (TEM) and far-UV circular dichroism. The chaperone-like activity of each of the heat-treated samples was measured using the DTT induced insulin aggregation assay. RESULTS: The native alpha-crystallin samples showed secondary structure perturbations, an increase in aggregate size and asymmetry, and an increase in chaperone-like activity after heat incubation above 50 degrees C. The other three sample types showed secondary structure perturbations beginning at lower incubation temperatures, and a progressive decrease in chaperone-like activity with exposure to increasing temperatures. TEM showed all samples formed large asymmetric high molecular weight aggregates after incubation at 65 degrees C. CONCLUSIONS: The urea denaturation/renaturation of alpha-crystallin has been shown to result in the loss of a small amount of alpha-helix, but to have no effect on chaperone-like activity under standard test conditions. The present results indicate this lost alpha-helix may be responsible for the differential effects of heat incubation on the different forms of alpha-crystallin.

Animals↗