Probing the three-dimensional structure of human calreticulin

Calreticulin (CRT) is an abundant soluble protein of the endoplasmic reticulum lumen that functions as a molecular chaperone for nascent glycoproteins. We have probed the three-dimensional structure of human CRT using a series of biochemical and biophysical approaches in an effort to understand the molecular basis of its chaperone function. Sedimentation analysis and chemical cross-linking experiments showed that CRT is monodisperse and monomeric in solution with a molecular mass (MW) of 46 +/- 1 kDa. This MW value together with a sedimentation coefficient, s(o)(20,w), of 2.71 S yielded a frictional ratio, f/f(0), of 1.65. Assuming CRT to be a prolate ellipsoid, we calculated an apparent length of 29.8 nm and diameter of 2.44 nm consistent with an asymmetric elongated molecule. These hydrodynamic dimensions account for the apparent anomalous elution position of CRT on gel filtration columns. Far-UV circular dichroism experiments showed that CRT has a cooperative thermal denaturation transition with a midpoint temperature of 42.5 degrees C suggesting a marginally stable structure. Proteolysis experiments showed that the highly acidic segment at the C-terminus of CRT is most susceptible to digest, consistent with the absence of a well-defined polypeptide backbone structure in this region of the protein. Temperature-dependent proteolysis with thermolysin revealed a stable core region within the N- and P-domains. A stable fragment encompassing most of the P-domain was also identified in the thermolytic mixture. Collectively, our results suggest that CRT is likely to be a flexible molecule in solution which may be important for its chaperone function.     

Probing of the secondary structure of maxizymes.

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. In order to probe the putative conformational changes, we used a weakly alkaline solution to hydrolyze differentially phosphodiester bonds that were located in different environments. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA.     

Probing the structure of the dimeric KtrB membrane protein

The KtrAB ion transporter is a complex of two proteins, KtrB and KtrA. The integral membrane protein KtrB is expected to adopt the structural architecture typified by the pore domain of potassium channels. Here we show that homo-dimerization of KtrB proteins is most likely a general property of this family of transporters. Using cysteine mutants and bifunctional cross-linkers we define regions of the Bacillus subtilis KtrB molecule that are close to the molecular 2-fold axis and to the dimer interface. Fitting of the cross-linking data to a potassium channel-like model suggests structural similarities between potassium channels and KtrB proteins in the extracellular half of the molecule and differences in the cytoplasmic regions.     

On the chemical constitution of cometary nuclei

The observed properties of comets and the experiments of Anderset al. suggest that during the condensation of the outer parts of the solar system from the solar nebula-assumed to contain most of the C in the form of CO, the remaining O mostly in the form of H₂O, and the excess H as H₂ molecules-besides the most stable hydrocarbon (CH₄) also considerable quantities of heavier hydrocarbon were formed. When the surface of the nucleus is warmed up in re-approaching the sun, part of the solar quanta having sufficient energy for dissociating such molecules, exothermic reactions, for instance such leading, to the observed C₂ and C₃ molecules may be expected; an inhomogeneous and porous structure of the nucleus would explain the observed occurrence of short-lived discrete jets, in which a small fraction of the CO could be ionized. Since also N must be present in some quantity (observed mainly was CN and N₂⁺), the formation of more complex organic molecules must also have taken place. To which extent complex interstellar molecules can have survived the formation of the solar system is hard to say on the basis of present knowledge of the origin of the solar system.     

Probing the structure of Lhca3 by mutation analysis

Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.     

Probing the structure of Lhca3 by mutation analysis

Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.     

Probing chromatin structure in the early phases of apoptosis

Abstract. A typical flow cytometric marker of apoptosis is the appearance of a hypodiploid peak. This phenomenon is related to the chromatin fragmentation and loss that occurs during the late stages of the process. We describe herein the changes occurring at the chromatin level in purified nuclei preparations obtained from human peripheral blood mononuclear cells in a time-course study, including the simultaneous evaluation of nuclear proteins and DNA stainability, light-scattering properties, and spectrophotometric determination of the protein content. An augmentation of fluoroscein isothiocyanate (FITC) stainability was noticed as early as 1 h after irradiation. As this phenomenon is not correlated to changes in actual protein content, one can conclude that modifications of basic protein accessibility occur from the early phases of the apoptotic process. Also DNA stainability augmented with time, generating the transient appearance of a hyperdiploid peak that preceded the appearance of the hypodiploid peak typical of the late stages of the process, and that shared with the latter the same light-scattering properties. Chromatin status was further explored by staining apoptotic nuclei using DNA probes with peculiar molecular weight. Propidium iodide (PI) and ethidium bromide (EB), but not the much bulkier 7-aminoactinomycin D (7-AAD), identified the nuclei with a transient increase in DNA stainability confirming that an increased dye accessibility to binding sites was responsible for the phenomenon. Remarkably, all dyes identified the same proportion of hypodiploid nuclei when an apoptotic nucleus shed its fragmented chromatin. Control experiments included differential interference contrast and fluorescence microscopy that showed the purity of nuclei preparations and the typical morphological apoptotic features. Finally, the simultaneous evaluation of DNA by PI and nuclear proteins by FITC in a time course study allowed a thorough assessment of changes occurring at the chromatin level in the diverse stages of apoptosis. It is suggested that proteolysis precedes endonucleolysis and probably renders it easier the final endonucleolytic step leading to DNA fragmentation and loss.     

Probing the structure and function of viral RNA genomes

The majority of human, animal and plant viral pathogens possess genomes composed of RNA. The strategies evolved for expression and replication of viral RNA genomes can differ significantly from those utilized for expression and replication of host-cell genetic material. Consequently, knowledge of the molecular details of these strategies can lead to a clearer understanding of the origin, evolution and control of viral pathogens. We describe recent progress in identifying important structural and functional domains of the RNA genomes and associated replicative enzymes for two very different viruses: vesicular stomatitis virus, which possesses a single-stranded RNA genome of negative polarity, and wound tumor virus, which contains a genome composed of 12 discrete segments of double-stranded RNA.     

Probing the structure and activity of trypsin with amidination

Trypsin reacts with S-methylisothiourea for 1 to 2 h and the number of primary amine sites at which covalent labeling occurs is determined by mass spectrometry. By digesting the amidinated trypsin and mass analyzing the proteolytic peptides the sites of reaction are determined. The addition of cytochrome c to a solution of amidinated trypsin enables the proteolytic activity and autolytic properties of the enzyme to be studied.     

Probing protein structure by limited proteolysis

Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, alpha-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to prompt its systematic use as a simple first step in the elucidation of structure-dynamics-function relationships of a novel and rare protein, especially if available in minute amounts.     

Probing the structure of photosystem II with amines and phenylhydrazine

Photosynthetic oxygen evolution is catalyzed at the manganese-containing active site of photosystem II (PSII). Amines are analogs of substrate water and inhibitors of oxygen evolution. Recently, the covalent incorporation of (14)C from [(14)C]methylamine and benzylamine into PSII subunits has been demonstrated (Ouellette, A. J. A., Anderson, L. B., and Barry, B. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-2209). To obtain more information concerning these labeling reactions, t-[(14)C]butylamine and phenylhydrazine were employed as probes. Neither compound can be oxidized by a transamination or addition/elimination mechanism, but both can react with activated carbonyl groups, produced as a result of posttranslational modification of amino acid residues, to give amine-derived adducts. (14)C incorporation into the PSII subunits D2/D1 and CP47 was obtained upon treatment of PSII with either t-[(14)C]butylamine or [(14)C]phenylhydrazine. For t-butylamine and methylamine, the amount of labeling increased when PSII was treated with denaturing agents. Labeling of CP47, D2, and D1 with methylamine and phenylhydrazine approached a one-to-one stoichiometry, assuming that D2 and D1 each have one binding site. Evidence was obtained suggesting that reductive stabilization and/or access are modulated by PSII light reactions. These results support the proposal that PSII subunits D2, D1, and CP47 contain quinocofactors and that access to these sites is sterically limited.     

Probing the structure and bifunctionality of catalase-peroxidase (KatG)

Catalase-peroxidases (KatGs) exhibit peroxidase and substantial catalase activities similar to monofunctional catalases. Crystal structures of four different KatGs reveal the presence of a peroxidase-conserved proximal and distal heme pocket together with features unique to KatG. To gain insight into their structure-function properties, many variants were produced and very similar results were obtained irrespective of the origin of the KatG mutated. This review focuses mainly on the electronic absorption and resonance Raman results together with the combined analysis of pre-steady and steady-state kinetics of various mutants involving both the peroxidase-conserved and the KatG-specific residues of recombinant KatG from the cyanobacterium Synechocystis. Marked differences in the structural role of conserved amino acids and hydrogen-bond networks in KatG with respect to the other plant peroxidases were found. Typically, the catalatic but not the peroxidatic activity was very sensitive to mutations that disrupted the KatG-typical extensive hydrogen-bonding network. Moreover, the integrity of this network is crucial for the formation of distinct protein radicals formed upon incubation of KatG with peroxides in the absence of one-electron donors. The correlation between the structural architecture and the bifunctional activity is discussed and compared with data obtained for KatGs from other organisms.     

Probing the structure and function of the nuclear pore complex.

Nucleocytoplasmic transport is a bi-directional process mediated by the nuclear pore complex (NPC), which results in a segregation of cytoplasmic and nuclear macromolecules within cells. Some progress has been made in understanding the mechanistic basis of this selective transport phenomenon. In particular, cryo-electron microscopy of frozen-hydrated nuclear envelopes coupled with image processing and labeling studies, has provided a glimpse of the transporter at the center of the NPC.     

Probing ribosome structure by europium-induced RNA cleavage

Divalent metal ions are absolutely required for the structure and catalytic activities of ribosomes. They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metal ion induced RNA cleavages are useful for characterising metal ion binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA of E. coli. The cleavage sites were identified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions. Several Eu3+ cleavage sites, mostly those at which a general metal ion binding site had been already identified, were identical to previously described divalent metal ions. Overall, the Eu3+ cleavages are most similar to the Ca2+ cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+ were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+ cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+ binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.     

Probing the functional importance of the hexameric ring structure of RNase PH

RNase PH is a phosphate-dependent exoribonuclease that catalyzes the removal of nucleotides at the 3' end of the tRNA precursor, leading to the release of nucleoside diphosphate, and generates the CCA end during the maturation process. The 1.9-A crystal structures of the apo and the phosphate-bound forms of RNase PH from Pseudomonas aeruginosa reveal a monomeric RNase PH with an alpha/beta-fold tightly associated into a hexameric ring structure in the form of a trimer of dimers. A five ion pair network, Glu-63-Arg-74-Asp-116-Arg-77-Asp-118 and an ion-pair Glu-26-Arg-69 that are positioned symmetrically in the trimerization interface play critical roles in the formation of a hexameric ring. Single or double mutations of Arg-69, Arg-74, or Arg-77 in these ion pairs leads to the dissociation of the RNase PH hexamer into dimers without perturbing the overall monomeric structure. The dissociated RNase PH dimer completely lost its binding affinity and catalytic activity against a precursor tRNA. Our structural and mutational analyses of RNase PH demonstrate that the hexameric ring formation is a critical feature for the function of members of the RNase PH family.     

Probing of DNA polymorphic structure in the cell with osmium tetroxide

It is shown that osmium tetroxide, 2,2'-bipyridine can be applied as a probe of DNA structure in a bacterial cell. Using this probe we demonstrate (a) presence of structural distortions at the junctions between the right-handed B and left-handed Z DNA in a recombinant plasmid pRW751 and (b) unusual structure of the d(A-T)16 insert in pAT32 plasmid in E. coli cells and in in vitro.