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R Jaenicke

Publications and source records attributed to R Jaenicke.

At least 73 records · Page 4Linked to original sources
PubMed1997

A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability.

Triosephosphate isomerase (TIM) is a very stable dimer. In order to understand better the importance of dimerization for stability and catalytic activity, we have constructed a monomeric double-mutation variant. The dimer interface residues Thr75 and Gly76, which are at the tip of loop 3, have been substituted by an arginine and a glutamate, respectively. In wild type TIM, these two residues are at a distance of 27 A from the active site (as measured within the same subunit). This new variant, RE-TIM, was expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Sedimentation equilibrium ultracentrifugation runs showed that RE-TIM is a monomer in solution. Far-UV CD spectra indicate that this new variant is folded properly and that the secondary structure contents of RE-TIM are similar to those of wild type TIM. The monomeric RE-TIM has residual TIM activity. The thermal stability of RE-TIM is lower than that for wild type TIM. CD melting curves for RE-TIM and wild type TIM show Tm values of 52 and 57 degrees C, respectively, in the presence of the active site ligand 2-phosphoglycolate at 1 mM. Previously, we have characterized two other monomeric forms of TIM: monoTIM and H47N-TIM. The properties of RE-TIM, H47N-TIM, and monoTIM are compared, and it is argued that the properties of RE-TIM will be very similar to those of wild type monomeric subunits. This implies that wild type monomeric subunits have some stability and are catalytically active. It is also inferred that these monomeric subunits have flexible loops which rigidify at the dimer interface on dimerization, causing a 1000-fold increase of kcat and a 10-fold decrease of Km.

Catalysis↗
PubMed1997

The domains in gammaB-crystallin: identical fold-different stabilities.

gammaB-crystallin from vertebrate eye lens is an all beta-sheet two-domain protein with a high degree of intrachain symmetry. Its N and C-terminal domains show high levels of sequence similarity and structural identity. In natural gammaB-crystallin, the domains fold independently. The recombinantly expressed isolated domains are stable monomeric proteins, which do not associate spontaneously to form a gammaB-like dimer. In contrast to their identical folding topology, the two domains obviously follow different folding mechanisms. While the two-state model is valid for the C-terminal domain, the folding behaviour of the N-terminal domain is more complex. The stability of the C-terminal domain is strongly dependent on pH. At pH 2, the C-terminal domain in its isolated form is significantly less stable than within the gammaB-molecule. In contrast, the isolated N-terminal domain does not differ in its stability from the N-terminal domain in wild-type gammaB-crystallin. The strongly decreased stability of the C-terminal domain at acid pH allowed a dissection of the intrinsic stabilities of the domains and their interactions in gammaB-crystallin. At pH 2, domain interactions contribute -16 kJ/mol to the overall stability of gammaB-crystallin.

Amino Acid Sequence↗
PubMed1997

Mutational analysis of hydrophobic domain interactions in gamma B-crystallin from bovine eye lens.

gamma B-crystallin is a monomeric member of the beta gamma-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-beta Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete gamma B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for gamma B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of gamma B-crystallin.

Animals↗
PubMed1997

The X-ray structure of a mutant eye lens beta B2-crystallin with truncated sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.

Amino Acid Sequence↗
PubMed1997

Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: structure and stability of the recombinant enzyme and its isolated cellulose-binding domain.

The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120-kDa endo-1,4-D-xylanase exhibiting high intrinsic stability and a temperature optimum approximately 90 degrees C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C-terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose-binding domain within the multi-domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431-444). In order to characterize the domain organization and the stability of XynA and its C-terminal cellulose-binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand-free form, was expressed as a glutathione S-transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield > 80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far-UV circular dichroism in order to characterize protein stability, guanidine-induced unfolding of XynA leads to biphasic transitions, with half-concentrations c1/2 (GdmCl) approximately 4 M and > 5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine-transition from 5 to 7 M GdmCl. This can be tentatively attributed to the cellulose-binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi-state behavior of XynA. In the case of CBD, a temperature-induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far-UV CD in the native state.

Amino Acid Sequence↗
PubMed1997

Dissection of the gene of the bifunctional PGK-TIM fusion protein from the hyperthermophilic bacterium Thermotoga maritima: design and characterization of the separate triosephosphate isomerase.

Triosephosphate isomerase (TIM), from the hyperthermophilic bacterium Thermotoga maritima, has been shown to be covalently linked to phosphoglycerate kinase (PGK) forming a bifunctional fusion protein with TIM as the C-terminal portion of the subunits of the tetrameric protein (Schurig et al., EMBO J 14:442-451, 1995). To study the effect of the anomalous state of association on the structure, stability, and function of Thermotoga TIM, the isolated enzyme was cloned and expressed in Escherichia coli, and compared with its wild-type structure in the PGK-TIM fusion protein. After introducing a start codon at the beginning of the tpi open reading frame, the gene was expressed in E.c.BL21(DE3)/ pNBTIM. The nucleotide sequence was confirmed and the protein purified as a functional dimer of 56.5 kDa molecular mass. Spectral analysis, using absorption, fluorescence emission, near- and far-UV circular dichroism spectroscopy were used to compare the separated Thermotoga enzyme with its homologs from mesophiles. The catalytic properties of the enzyme at approximately 80 degrees C are similar to those of its mesophilic counterparts at their respective physiological temperatures, in accordance with the idea that under in vivo conditions enzymes occupy corresponding states. As taken from chaotropic and thermal denaturation transitions, the separated enzyme exhibits high intrinsic stability, with a half-concentration of guanidinium-chloride at 3.8 M, and a denaturation half-time at 80 degrees C of 2 h. Comparing the properties of the TIM portion of the PGK-TIM fusion protein with those of the isolated recombinant TIM, it is found that the fusion of the two enzymes not only enhances the intrinsic stability of TIM but also its catalytic efficiency.

Bacterial Proteins↗
PubMed1997

Protein engineering with monomeric triosephosphate isomerase (monoTIM): the modelling and structure verification of a seven-residue loop.

Protein engineering experiments have been carried out with loop-1 of monomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is disordered in every crystal structure of liganded monoTIM, but in the wild-type TIM it is a very rigid dimer interface loop. This loop connects the first beta-strand with the first alpha-helix of the TIM-barrel scaffold. The first residue of this loop, Lys13, is a conserved catalytic residue. The protein design studies with loop-1 were aimed at rigidifying this loop such that the Lys13 side chain points in the same direction as seen in wild type. The modelling suggested that the loop should be made one residue shorter. With the modelling package ICM the optimal sequence of a new seven-residue loop-1 was determined and its structure was predicted. The new variant could be expressed and purified and has been characterized. The catalytic activity and stability are very similar to those of monoTIM. The crystal structure (at 2.6 A resolution) shows that the experimental loop-1 structure agrees well with the modelled loop-1 structure. The direct superposition of the seven loop residues of the modelled and experimental structures results in an r.m.s. difference of 0.5 A for the 28 main chain atoms. The good agreement between the predicted structure and the crystal structure shows that the described modelling protocol can be used successfully for the reliable prediction of loop structures.

Amino Acid Sequence↗
PubMed1997

The PGK-TIM fusion protein from Thermotoga maritima and its constituent parts are intrinsically stable and fold independently.

In the hyperthermophilic bacterium Thermotoga maritima, the two glycolytic enzymes phosphoglycerate kinase (PGK) and triosephosphate isomerase (TIM) are covalently connected forming a tetrameric single-chain PGK-TIM fusion protein. A frameshift allows the translation of PGK alone, whereas TIM activity exclusively resides in the fusion protein (Schurig et al., 1995). Cloning the pgk-tim gene from Thermotoga maritima in Escherichia coli, yields monomeric PGK and tetrameric PGK-TIM fusion protein as authentic recombinant proteins (Beaucamp et al., 1995). Both exhibit high intrinsic stability. The thermal transitions at approximately 80 degrees C are irreversible, rendering determination of thermodynamic data impossible. The half-concentrations, (cGdmCl)1/2, of the guanidinium-chloride induced unfolding transitions are 3.0 and 3.9 M GdmCl for PGK and the PGK-TIM fusion protein, respectively. Monitoring denaturation by activity, fluorescence emission and circular dichroism, deactivation and unfolding of the two-domain PGK is found to precede the transitions of the TIM domain. With increasing temperature, (cGdmCl)1/2 is shifted to lower denaturant concentrations; at the same time, the transitions change from bimodal to unimodal. As indicated by the incomplete reversibility of the deactivation/unfolding/dissociation transitions, misfolding, as well as wrong domain interactions seem to interfere with the correct folding and association of the bienzyme complex.

Bacterial Proteins↗
PubMed1997

Determination of the regulatory disulfide bonds of NADP-dependent malate dehydrogenase from Pisum sativum by site-directed mutagenesis.

The light-mediated reversible activation of NADP-dependent malate dehydrogenase (NADP-MDH) from Pisum sativum can be simulated in vitro by reducing the inactive oxidized enzyme with dithiothreitol. Since the gross structure and the dimeric state of the enzyme are unaffected by the state of oxidation, the redox modulation cannot be attributed to inter-subunit disulfide bridges. In order to identify intra-chain cystine cross bridges that might be candidates responsible for the activation reaction, site-directed mutagenesis experiments were performed, substituting alanine for up to four exposed cysteine residues. Mutants were expressed in freshly transformed EcoB cells and purified to homogeneity. As indicated by the activation behavior (by dithiothreitol-mediated thioldisulfide exchange), disulfides C23-C28 in the N-terminal and C364-C376 in the C-terminal part of the polypeptide chain are involved in the light-induced modulation of the activity of the wild type enzyme. A mutant of the enzyme lacking the N-terminal 45 residues confirms this result. Electrophoretic mobility and FPLC prove the wild type enzyme and its mutants to be dimeric; differences refer to the packing of the N- and C-terminal portions of the enzyme in its oxidized and reduced state. The kinetics of the redox modulation differ, depending on the solvent conditions and the mode of activation. After elimination of the N-terminal disulfide bond, sigmoidal activation profiles are no longer observed, suggesting a slow conformational rearrangement in the N-terminal portion of the wild type enzyme to be rate-limiting in the course of reductive activation. For the wild type, this finding can be mimicked in the presence of non-denaturing concentrations of guanidinium-chloride.

Alanine↗
PubMed1996

Tetrameric and octameric lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. Structure and stability of the two active forms.

Lactate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been functionally expressed in Escherichia coli. As shown by gel-permeation chromatography, dynamic light scattering, and ultracentrifugation, the recombinant protein forms homotetrameric and homooctameric assemblies with identical spectral properties and a common subunit molecular mass (35 kDa). Dynamic light scattering and sedimentation equilibrium experiments proved that both species are monodisperse, thus excluding their interconversion in the given ranges of concentration (0.02-50 mg/ml) and temperature (20-80 degrees C). Rechromatography confirms this finding: the octamer does not dissociate at low enzyme concentrations, nor do tetramers dimerize at the given upper limit of concentration. Renaturation of pure tetramers or octamers after preceding guanidine denaturation leads to redistribution of the two species; increased temperature favors octamer formation. Thermal analysis and denaturation by chaotropic agents do not allow the free energies of stabilization of the two forms to be quantified, because heat coagulation and kinetic partitioning between reconstitution and aggregation causes irreversible side reactions. Guanidine denaturation of the octamer leads to a highly cooperative dissociation to tetramers which subsequently dissociate and unfold to yield metastable dimers and, finally, fully unfolded monomers. Evidently, there is no tight coupling of the two tetramers within the stable octameric quaternary structure. Electron microscopy clearly corroborates this conclusion: image processing shows that the dumb-bell-shaped octamer is made up of two tetramers connected via surface contacts without significant changes in the dimensions of the constituent parts.

Binding Sites↗
PubMed1996

Extremely thermostable L(+)-lactate dehydrogenase from Thermotoga maritima: cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state.

L(+)-lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195-201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 degrees C, i.e., ca. 60 degrees below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 degrees C show satisfactory diffraction suitable for X-ray analysis up to a resolution of 2.8 A. As shown by gel-permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near- and far-UV CD) of the two species are indistinguishable. The calculated alpha-helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unalatered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 degrees C; irreversible thermal denaturation becomes significant at approximately 95 degrees C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat.

Amino Acid Sequence↗
PubMed1996

How do proteins acquire their three-dimensional structure and stability?

Proteins are multifunctional in the sense that their specific amino acid sequence simultaneously determines self-organization, function and turnover. Correspondingly, evolution has to compromise between rigidity (stability) and flexibility (function/degradation) to the effect that the free energy of stabilization of proteins is the equivalent of only a few weak interactions (delta Gstab = 45 +/- 15 kJ.mol-1). Molecular adaptation of thermophiles, psychrophiles and other extremophiles is accomplished by extrinsic factors that are not encoded in the amino acid sequence, or by minute local structural changes involving mainly ion pairs and hydrophobic side chains. The acquisition of the native three-dimensional structure may be described by single- or multiple-pathway folding and association, where the fast collapse of the polypeptide chain leads to molten-globule-like states; subsequent shuffling reactions yield structured monomers which, in the case of oligomers, undergo specific association to form the native functional state. The rate-limiting steps (cysteine oxidation, proline isomerization, subunit assembly) are catalyzed or directed by enzymes or chaperones.

Calorimetry↗
PubMed1996

Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization.

Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.

Amino Acid Sequence↗
PubMed1996

The X-ray structures of two mutant crystallin domains shed light on the evolution of multi-domain proteins.

We use protein engineering and crystallography to simulate aspects of the early evolution of beta gamma-crystallins by observing how a single domain oligomerizes in response to changes in a sequence extension. The crystal structure of the C-terminal domain of gamma beta-crystallin with its four-residue C-terminal extension shows that the domain does not form a symmetric homodimer analogous to the two-domain pairing in beta gamma-crystallins. Instead the C-terminal extension now forms heterologous interactions with other domains leading to the solvent exposure of the natural hydrophobic interface with a consequent loss in protein solubility. However, this domain truncated by just the C-terminal tyrosine forms a symmetric homodimer of domains in the crystal lattice.

Amino Acid Sequence↗
PubMed1996

The elusive role of the N-terminal extension of beta A3- and beta A1-crystallin.

beta-Crystallins are structural lens proteins with a conserved two-domain structure and variable N- and C-terminal extensions. These extensions are assumed to be involved in quaternary interactions within the beta-crystallin oligomers or with other lens proteins. Therefore, the production of beta A3- and beta A1-crystallin from the single beta A3/A1 mRNA by dual translation initiation is of interest. These crystallins are identical, except that beta A1 has a much shorter N-terminal extension that beta A3. This rare mechanism has been conserved for over 250 million years during the evolution of the beta A3/A1 gene, suggesting that the generation of different N-terminal extensions confers a selective advantage. We therefore compared the stability and association behaviour of recombinant beta A3- and beta A1-crystallin. Both proteins are equally stable in urea- and pH-induced denaturation experiments. Gel filtration and analytical ultracentrifugation established that beta A3 and beta A1 both form homodimers. In the water-soluble proteins of bovine lens, beta A3 and beta A1 are present in the same molecular weight fractions, indicating that they oligomerize equally with other beta-crystallins. 1H-NMR spectroscopy showed that residues Met1 to Asn22 of the N-terminal extension of beta A3 have great flexibility and are solvent exposed, excluding them from protein interactions in the homodimer. These results indicate that the different N-terminal extensions of beta A3 and beta A1 do not affect their homo- or heteromeric interactions.

Amino Acid Sequence↗