Biomedical subjects

R Jaenicke

Publications and source records attributed to R Jaenicke.

At least 19 recordsLinked to original sources

PubMed1995

Kinetics of translation of gamma B crystallin and its circularly permutated variant in an in vitro cell-free system: possible relations to codon distribution and protein folding.

Analysis of nascent gamma B-crystallin peptides accumulating during in vitro translation in a rabbit reticulocyte lysate cell-free system was carried out. As a consequence of the irregular distribution of rare codons along the polypeptide chain of gamma B-crystallin, translation of the two-domain protein is a non-uniform process characterized by specific pauses. One of the major delays occurs during the translation of the connecting peptide between the domains. Comparing the kinetics of translation of natural gamma B-crystallin and its circularly permutated variant (with the order of the N- and C-terminal domains exchanged) reveals that the natural N-terminal domain is translated faster than the C-terminal one. Since the N-terminal domain in natural gamma B-crystallin is known to be more stable and to fold faster than the C-terminal one [E.-M. Mayr et al. (1994) J. Mol. Biol. 235, 84-88], the present data suggest that the translation rates are optimized to tune the synthesis and folding of the nascent polypeptide chain. In this connection, the pause in the linker region between the domains provides a delay allowing the correct folding of the N-terminal domain and its subsequent assistance in the stabilization of the C-terminal one.

Animals↗

PubMed1995

An interface point-mutation variant of triosephosphate isomerase is compactly folded and monomeric at low protein concentrations.

Wild-type trypanosomal triosephosphate isomerase (wtTIM) is a very tight dimer. The interface residue His-47 of wtTIM has been mutated into an asparagine. Ultracentrifugation data show that this variant (H47N) only dimerises at protein concentrations above 3 mg/ml. H47N has been characterised at a protein concentration where it is predominantly a monomer. Circular dichroism measurements in the near-UV and far-UV show that this monomer is a compactly folded protein with secondary structure similar as in wtTIM. The thermal stability of the monomeric H47N is decreased compared to wtTIM: temperature gradient gel electrophoresis (TGGE) measurements give Tm-values of 41 degrees C for wtTIM, whereas the Tm-value for the monomeric form of H47N is approximately 7 degrees C lower.

Animals↗

PubMed1995

Structure of human parathyroid hormone 1-37 in solution.

Human parathyroid hormone (hPTH), amino acids Ser1 to Leu37, is biologically active with respect to both receptor binding and activation of adenylate cyclase to influence the serum calcium concentration. It induces DNA synthesis via an unknown signal pathway. We investigated the structure of hPTH(1-37) in H2O/buffer solution under near physiological conditions, that is pH 6.0 and 270 mM salt, by circular dichroism, ultracentrifugation, nuclear magnetic resonance spectroscopy, and molecular dynamics calculations. Complete sequence specific assignments of all 1H resonances were performed by using 1H two-dimensional NMR measurements (double quantum-filtered correlated spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total correlation spectroscopy with suppression of NOESY-type cross-peaks spectra). hPTH(1-37) obtained helical structure and showed hydrophobic interactions defining a tertiary structure. The NH2-terminal four amino acids of hPTH(1-37) did not show a stable conformation. Evidence for an alpha-helical region between Ile5 and Asn10 was found. This region was followed by a flexible link (Gly12, Lys13) and a well defined turn region, His14 to Ser17. The latter was stabilized by hydrophobic interactions between Trp23 and Leu15. Ser17 through at least Leu28 formed an alpha-helix. Arg20 and Lys27 were involved in the core built by His14 to Ser17. Unrestrained molecular dynamics simulations indicated that the structure was stable on the 200 ps time scale.

Amino Acid Sequence↗

PubMed1995

Folding and association versus misfolding and aggregation of proteins.

The acquisition of spatial structure in proteins may be described in terms of hierarchical condensation, with contributions of local interactions between next neighbours and the interactions between domains and subunits accumulating to create the marginal free energy of stabilization characteristic of the functional state of globular proteins. Domains represent independent folding units such that the overall kinetics divide into the sequential collapse of subdomains and domains and their merging to form the compact tertiary structure. In proceeding to oligomeric proteins, docking of subunits follows the formation of structured monomers. Thus, the overall mechanism of folding and association obeys consecutive uni-bimolecular kinetics. Beyond a limiting protein concentration, aggregation will outrun proper domain pairing and subunit association. In the cell, accessory proteins are involved in catalysis of the rate-determining steps of folding (proline isomerization and SH-SS exchange) and in the kinetic partitioning between folding and aggregation (chaperone action). The practical aspects of accessory proteins have been investigated in detail using immunotoxins and antibody fragments as examples. Additional concepts allowing off-pathway reactions in protein reconstruction to be kept to a minimum refer to pulse-dilation, reverse micelles and immobilization of polypeptide chains on matrices.

Kinetics↗

PubMed1995

The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima at 2.5 A resolution.

The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophile Thermotoga maritima was determined by Patterson search methods using the known structure of the Bacillus stearothermophilus enzyme. The structure was refined at a resolution of 2.5 A to an R-factor of 16.63% for 26289 reflections between 8.0 A an 2.5 A with F > 2 sigma(F). The crystallographic asymmetric unit contains two monomers related by approximate 2-fold symmetry and a tetramer is built up by crystallographic symmetry. The root-mean-square deviation of Ca positions of glyceraldehyde-3-phosphate dehydrogenase from T. maritima and B. stearothermophilus is 0.83 A in the NAD+ binding domains and smaller close to the cofactor. In contrast, the largest deviations in the catalytic domains are found at residues involved in coordination of sulphate ion SO4 339, which most likely marks the site of the attacking inorganic phosphate ion in catalysis. A large number of extra salt-bridges may be an important factor contributing to the high thermostability of this protein.

Amino Acid Sequence↗

PubMed1995

Phosphoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex.

Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and identical N-terminal sequence was also found. Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T. maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far. The corresponding pgk and tpi genes are part of the apparent gap operon of T. maritima. This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene. On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.

Amino Acid Sequence↗

PubMed1995

Octameric enolase from the hyperthermophilic bacterium Thermotoga maritima: purification, characterization, and image processing.

Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.

Amino Acid Sequence↗

PubMed1995

Characterization of a quaternary-structured folding intermediate of an antibody Fab-fragment.

Antibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain-domain interactions lead to a rate-limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse (kappa/IgG1). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late-folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native-like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weekly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a native-like structure, follows a two-step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.

Acrylamides↗

PubMed1994

Dissociation and reconstitution of the Thermoplasma proteasome.

The proteasome from the thermoacidophilic archaeon Thermoplasma acidophilum in its native state represents a 20S particle with significant secondary structure (approximately 35% alpha helix) of its subunits. Electron microscopy, ultracentrifugal and spectral analysis demonstrate that at pH of less than 3 dissociation to partially denatured subunits occurs. Upon dialysis against near neutral pH buffers, at low protein concentration, reconstitution occurs, leading to the restoration of up to 90% of the native fluorescence signal. The recovery of activity depends on several parameters, including the buffer system, the pH used to dissociate the complex, and the duration of exposure to low pH. High concentrations of Ca2+ and Mg2+ cause partial dissociation of the Thermoplasma proteasome, yielding distinct subcomplexes. Neither the completely nor the partially dissociated complexes have proteolytic activity, indicating that function is linked to fully assembled proteasomes.

Circular Dichroism↗

PubMed1994

Symmetric complexes of GroE chaperonins as part of the functional cycle.

The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

Adenosine Diphosphate↗

PubMed1994

Erythrina caffra trypsin inhibitor retains its native structure and function after reducing its disulfide bonds.

Erythrina trypsin inhibitor (ETI) from the seeds of Erythrina caffra is a high-affinity inhibitor of trypsin, chymotrypsin and tissue plasminogen activator. Its 172 amino acid polypeptide chain is stabilized in its compact, native state by two disulfide bonds. In spite of their conservation in all trypsin inhibitors of the soybean trypsin inhibitor (STI-Kunitz) family, their state of oxidation is essential only for protein stability but not for inhibitory function. Reduction/reoxidation of ETI in the presence of glutathione reshuffling buffer (GSH/GSSG; pH 8.3) not only allows the inhibitor to be restored in its native structure, but also does not interfere with its binding affinity; carboxymethylation or carboxamidomethylation of the free thiol groups does not affect K1 significantly (for trypsin (KI)ETIox = 2.3 nM, (KI)ETICM = 1.9 nM; for chymotrypsin (KI)ETIox = 30 microM, (KI)ETICM = 25 microM). The two cystine cross-bridges in the native ETI lead to enhanced stability toward pH and chaotropic agents. As taken from intrinsic protein fluorescence at acid pH and varying ionic strength (pH < 4, I = 0.01 to 0.15 M), the oxidized inhibitor retains its spectral properties, whereas reduced and carboxymethylated or carboxamidomethylated ETI undergo at least partial denaturation. At alkaline pH, the oxidized protein is stable up to pH 9.5, whereas the reduced protein undergoes structural alterations at pH > 7, reaching a final plateau at pH 10.0 to 10.5. In the case of urea (U) or guanidinium chloride (GdmCl) denaturation at pH 7.0, structural transitions of the oxidized inhibitor show "hysteresis" with half-concentrations (cU)1/2 approximately 10 M and (cGdmCl)1/2 approximately 4.5 M for denaturation, and (cU)1/2 = 4.7 M and (cGdmCl)1/2 = 1.5 M for renaturation. In contrast, the reduced (and chemically modified) inhibitors exhibit true equilibrium transitions at (cU)1/2 = 0.9 M and (cGdmCl)1/2 = 0.5 M, respectively. Reduction/reoxidation in the absence and in the presence of denaturants (GdmCl) can also be applied to ETI covalently attached to a solid matrix.

Chymotrypsin↗

PubMed1994

Proteins under pressure. The influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes.

Oceans not only cover the major part of the earth's surface but also reach into depths exceeding the height of the Mt Everest. They are populated down to the deepest levels (approximately 11,800 m), which means that a significant proportion of the global biosphere is exposed to pressures of up to 120 MPa. Although this fact has been known for more than a century, the ecology of the 'abyss' is still in its infancy. Only recently, barophilic adaptation, i.e. the requirement of elevated pressure for viability, has been firmly established. In non-adapted organisms, increased pressure leads to morphological anomalies or growth inhibition, and ultimately to cell death. The detailed molecular mechanism of the underlying 'metabolic dislocation' is unresolved. Effects of pressure as a variable in microbiology, biochemistry and biotechnology allow the structure/function relationship of proteins conjugates to be analyzed. In this context, stabilization by cofactors or accessory proteins has been observed. High-pressure equipment available today allows the comprehensive characterization of the behaviour of proteins under pressure. Single-chain proteins undergo pressure-induced denaturation in the 100-MPa range, which, in the case of oligomeric proteins or protein assemblies, is preceded by dissociation at lower pressure. The effects may be ascribed to the positive reaction volumes connected with the formation of hydrophobic and ionic interactions. In addition, the possibility of conformational effects exerted by moderate, non-denaturing pressures, and related to the intrinsic compressibility of proteins, is discussed. Crystallization may serve as a model reaction of protein self-organization. Kinetic aspects of its pressure-induced inhibition can be described by a model based on the Oosawa theory of molecular association. Barosensitivity is known to be correlated with the pressure-induced inhibition of protein biosynthesis. Attempts to track down the ultimate cause in the dissociation of ribosomes have revealed remarkable stabilization of functional complexes under pseudo-physiological conditions, with the post-translational complex as the most pressure-sensitive species. Apart from the key issue of barosensitivity and barophilic adaptation, high-pressure biochemistry may provide means to develop new approaches to nonthermic industrial processes, especially in the field of food technology.

Bacterial Proteins↗

PubMed1994

Design, creation, and characterization of a stable, monomeric triosephosphate isomerase.

Protein engineering on trypanosomal triosephosphate isomerase (TIM) converted this oligomeric enzyme into a stable, monomeric protein that is enzymatically active. Wild-type TIM consists of two identical subunits that form a very tight dimer involving interactions of 32 residues of each subunit. By replacing 15 residues of the major interface loop by another 8-residue fragment, a variant was constructed that is a stable and monomeric protein with TIM activity. The length, sequence, and conformation of the designed fragment were suggested by extensive modeling.

Amino Acid Sequence↗

PubMed1994

Domain interactions and connecting peptides in lens crystallins.

beta B2- and gamma B-crystallin from bovine eye-lens are closely related proteins, topologically distinct mainly by virtue of the linker peptide connecting the two domains in each polypeptide chain. In homodimeric beta B2-crystallin, the extended conformation of the connecting peptide has been suggested to force the beta B2-molecule to favor intermolecular domain interactions compared with intramolecular contacts in monomeric gamma B-crystallin. From this one may postulate that the conserved interdomain contacts are essential for the overall stability of crystallins. This was clearly confirmed for gamma B-crystallin, since its isolated C-terminal domain is significantly less stable than in the context of native gamma B. Exchanging the linker peptide of gamma B- for that of beta B2-crystallin yields a monomeric protein with stability characteristics identical to gamma B-crystallin. We conclude that the domain-interface itself rather than the connecting peptide determines the mode of domain association in crystallins, as the linker in the gamma B beta-mutant is evidently twisted to a turn similar to the one in natural gamma B-crystallin.

Amino Acid Sequence↗

PubMed1994

Response of bacteria and fungi to high-pressure stress as investigated by two-dimensional polyacrylamide gel electrophoresis.

In an attempt to generalize previous observations (Jaenicke et al., Appl. Environ. Microbiol. 1988, 54, 2375-2380) and to find a convenient model system for studies of the pressure response, we tested the suitability of Escherichia coli and Thermotoga maritima (bacteria), and of five different eukaryotic species including the filamentous fungi Asteromyces cruciatus and Dendryphiella salina, and the marine yeasts Debaryomyces hansenii, Rhodosporidium sphaerocarpum, and Rhodotorula rubra. Using two-dimensional polyacrylamide gel electrophoresis, detailed investigations on the pressure response were carried out with E. coli and Rhodosporidium sphaerocarpum. In the former organism, major pressure response proteins could not be detected, although there are significant differences in expression of some proteins as well as some minor components that are found in all of the high pressure cell extracts but not in extracts from cultures grown at atmospheric pressure. In Rhodosporidium sphaerocarpum, no change in protein expression patterns was observed between 0.1 and 20 MPa. However, approaching the limit of viability of 50 MPa, additional protein spots became detectable at 45 MPa. This finding correlates with the observation of abnormal growth forms of the organism at this pressure (Lorenz, R. et al. manuscript in preparation).

Bacterial Proteins↗

PubMed1994

Autonomous folding of the excised coenzyme-binding domain of D-glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima.

An important question in protein folding is whether compact substructures or domains are autonomous units of folding and assembly. The protomer of the tetrameric D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has a complex coenzyme-binding domain, in which residues 1-146 form a compact substructure with the last 31 residues (313-333). Here it is shown that the gene of a single-chain protein can be expressed in Escherichia coli after deleting the 163 codons corresponding to the interspersed catalytic domain (150-312). The purified gene product is a soluble, monomeric protein that binds both NAD+ and NADH strongly and possesses the same unfolding transition induced by guanidinium chloride as the native tetramer. The autonomous folding of the coenzyme-binding domain has interesting implications for the folding, assembly, function, and evolution of the native enzyme.

Amino Acid Sequence↗

PubMed1994

Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions.

beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L). In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC). The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.

Amino Acid Sequence↗

PubMed1994

Three-dimensional model and quaternary structure of the human eye lens protein gamma S-crystallin based on beta- and gamma-crystallin X-ray coordinates and ultracentrifugation.

A 3-dimensional model of the human eye lens protein gamma S-crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3-dimensional structure of gamma-crystallin and beta-crystallin. The model is biased toward the monomeric gamma B-crystallin, which is more similar in sequence. Bovine gamma S-crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric gamma-crystallins. Because the linker has 1 extra residue compared with gamma B and beta B2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. gamma S-crystallin differs from gamma B-crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both beta- and gamma-crystallins, and may contribute to the different roles of these proteins in the lens.

Amino Acid Sequence↗