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R Jaenicke

Publications and source records attributed to R Jaenicke.

At least 127 records · Page 7Linked to original sources
PubMed1993

The catalytic activities of monomeric enzymes show complex pressure dependence.

High hydrostatic pressures in the biologically relevant range (< or = 1,200 bar) are known to cause dissociation of oligomeric enzymes in vitro, whereas protein denaturation requires pressures far beyond this range. Pressure-induced inactivation phenomena attributable to neither of these effects are shown to occur in monomeric enzymes. Three different types of pressure dependence can be distinguished: (1) a linear dependence of catalytic rate constants on pressure, as predicted by the activated complex theory, observed for lysozyme and thermolysin; (2) a biphasic profile consisting of two linear contributions, found for trypsin; (3) maximum curves, as observed for both directions of the octopine dehydrogenase reaction. The third case may be ascribed to a pressure-induced decrease in the partial specific volume of the protein, resulting in reduced flexibility of the active site. This mechanism may also apply to the pressure-induced inactivation of assembly systems stabilized against dissociation in the cell.

Amino Acid Oxidoreductases↗
PubMed1993

What does protein refolding in vitro tell us about protein folding in the cell?

The classical in vitro denaturation-renaturation studies by Anson, Anfinsen, Neurath, Pauling and others clearly suggested that the primary structure of proteins determines all higher levels of protein structure. Protein folding in the cell is inaccessible to a detailed analysis of its kinetic mechanism. There are obvious differences: nascent proteins acquire their native structure co- and post-translationally, with half-times in the minutes range, whereas refolding starts from the complete polypeptide chain, with rates varying from seconds to days. In the cell, accessory proteins are involved in regulating the rate of folding and association. Their role can be analysed both in vivo, by mutant studies, or by coexpression together with recombinant model proteins, and in vitro, by folding experiments in the absence and in the presence of 'foldases' and molecular chaperones, with the following general results: (i) folding is a sequential process involving native-like structural elements and a 'collapsed state' as early intermediates; (ii) the major side-reaction is caused by 'kinetic partitioning' between correct folding and wrong aggregation; (iii) rate-determining steps may be assisted by protein disulphide isomerase, peptidyl prolyl-cis-transisomerase, and molecular chaperones; and (iv) extrinsic factors, not encoded in the amino acid sequence, may be of crucial importance.

Amino Acid Isomerases↗
PubMed1993

The low-temperature folding intermediate of hyperthermophilic D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima shows a native-like cooperative unfolding transition.

Hyperthermophilic D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima, after denaturation in 6 M guanidinium chloride and subsequent renaturation by dilution at 3 degrees C, forms a 'low-temperature intermediate' with its native quaternary structure and most of its dichroic absorption restored, but with significant differences in its fluorescence properties compared to those of the native enzyme. Shifting the temperature beyond 10 degrees C, the enzyme is reconstituted to high yields and to an overall structure indistinguishable from the initial native state [FEBS Lett. 290 (1991) 235-238]. These criteria suggest that the cold intermediate represents an 'assembled molten globule'. However, present equilibrium transition data prove the cold intermediate to be native-like, in that it exhibits a reversible highly cooperative conformational transition to the unfolded state which is incompatible with the typical characteristics of the molten globule state of globular proteins.

Circular Dichroism↗
PubMed1993

Stabilization of creatinase from Pseudomonas putida by random mutagenesis.

Creatinase (creatine amidinohydrolase, EC 3.5.3.3) from Pseudomonas putida is a homodimer of 45 kDa subunit molecular mass, the three-dimensional structure of which is known at 1.9 A resolution. Three point mutants, A109V, V355M, and V182I, as well as one double mutant combining A109V and V355M, and the triple mutant with all three replacements, were compared with wild-type creatinase regarding their physical and enzymological properties. High-resolution crystal data for wild-type creatinase and the first two mutants suggest isomorphism at least for these three proteins (R. Huber, pers. comm.). Physicochemical measurements confirm this prediction, showing that the mutations have no effect either on the quaternary structure and gross conformation or the catalytic properties as compared to wild-type creatinase. The replacement of V182 (at the solvent-exposed end of the first helix of the C-terminal domain) does not cause significant differences in comparison with the wild-type enzyme. The other point mutations stabilize the first step in the biphasic denaturation transition without affecting the second one. In sum, the enhanced stability seems to reflect slight improvements in the local packing without creating new well-defined bonds. The increase in hydrophobicity generated by the introduction of additional methyl groups (A109V, V182I) must be compensated by minor readjustments of the global structure. Secondary or quaternary interactions are not affected. In going from single to double and triple mutants, to a first approximation, the increments of stabilization are additive.

Cloning, Molecular↗
PubMed1993

Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.

A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Reactivators↗
PubMed1993

Structural relationships of homologous proteins as a fundamental principle in homology modeling.

Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledge-based approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict.

Bacterial Proteins↗
PubMed1993

Effects of metal ions, thiamine diphosphate analogues and subunit interactions on the reconstitution behaviour of pyruvate decarboxylase from brewer's yeast.

The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.

Binding Sites↗
PubMed1993

Intrinsic stability and extrinsic stabilization of creatinase from Pseudomonas putida.

Creatinase (creatine amidinohydrolase, EC 3.5.3.3), a homodimer of 45 kDa subunit molecular mass, shows only limited functional stability, and is inaccessible to reconstitution after preceding deactivation, denaturation and dissociation. The enzyme has been characterized regarding its native and denatured states. Studying its unfolding characteristics in the presence of "extrinsic factors", such as DTE, BSA and glycerol, it was possible to define solvent conditions where the stability of the enzyme is significantly improved. Apart from protecting essential thiol groups and charge screening effects, the stabilization is caused mainly by preferential solvation. In the presence of 20% (w/v) glycerol, the kinetic analysis of the time course of denaturation indicates that a partially active folding intermediate, rather than the whole molecule, is involved in the stabilization. The mixed solvent improves the thermal stability, as well as the stability toward GdmCl and urea.

Circular Dichroism↗
PubMed1992

Glycosylation inhibits the interaction of invertase with the chaperone GroEL.

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.

Bacterial Proteins↗
PubMed1992

Stability and reconstitution of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima.

The molecular basis of thermal stability of globular proteins is a highly significant yet unsolved problem. The most promising approach to its solution is the investigation of the structure-function relationship of homologous enzymes from mesophilic and thermophilic sources. In this context, D-glyceraldehyde-3-phosphate dehydrogenase has been the most extensively studied model system. In the present study, the most thermostable homolog isolated so far is described with special emphasis on the stability of the enzyme under varying solvent conditions. D-Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima is an intrinsically thermostable enzyme with a thermal transition temperature around 110 degrees C. The amino acid sequence, electrophoresis, and sedimentation analysis prove the enzyme to be a homotetramer with a gross structure similar to its mesophilic counterparts. The enzyme in the absence and in the presence of its coenzyme, NAD+, exhibits no drastic structural differences except for a 3% change in sedimentation velocity reflecting slight alterations in the quaternary structure of the enzyme. At low temperature, in the absence of denaturants, neither "cold denaturation" nor subunit dissociation are detectable. Guanidinium chloride and pH-dependent deactivation precede the decrease in fluorescence emission and ellipticity, suggesting a complex denaturation mechanism. An up to 3-fold activation of the enzyme at low guanidinium concentration may be interpreted in terms of a compensation of the tight packing of the thermophilic enzyme at low temperature. Under destabilizing conditions, e.g. moderate concentrations of chaotropic agents, low temperature favors denaturation. The effect becomes important in reconstitution experiments after preceding guanidinium denaturation; the reactivation yield at low temperature drops to zero, whereas between 35 and 80 degrees C reactivation exceeds 80%. Shifting the temperature from approximately 0 degrees C to greater than or equal to 30 degrees C releases a trapped tetrameric intermediate in a fast reaction. Concentration-dependent reactivation experiments prove renaturation of the enzyme to involve consecutive folding and association steps. Reconstitution at room temperature yields the native protein, in spite of the fact that the temperature of the processes in vitro and in vivo differ by more than 60 degrees C.

Enzyme Activation↗
PubMed1992

Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.

Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1992

Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.

Enzyme Reactivators↗
PubMed1992

Correlation functions as a tool for protein modeling and structure analysis.

Proteins present unique folding structures whose conformations are determined primarily by their amino acid sequences. At present, there is no algorithm that would correlate the sequences with the structures determined by X-ray analysis or NMR. Comparative modeling of a new protein sequence based on the known structure of a functionally related protein promises to yield model structures that may provide relevant properties of the protein. To analyze the quality of a model structure, a set of correlation functions was derived from calculations on a subset of proteins from the structure database. Twenty-three highly resolved protein structures with resolutions of at least 1.7 A from various protein families were used as the primary database. The purpose of this initial work was to find highly sensitive functions (including statistical error limits for the parameters) that describe properties of "real" proteins. Each correlation described is characterized by the correlation coefficient, the parameters for linear or nonlinear regression (coefficients of the equation), standard deviation and variance, and the confidence limits describing the statistical probability for values to occur within these limits, e.g., the natural variability of the property under examination. In addition, a method was developed for creating reasonably misfolded proteins. The ability of a correlation function to discriminate between the native structure and the misfolded conformations is expressed by the reliability index, which indicates the sensitivity of a correlation function. The term correlation functions thus summarizes a variety of efforts to find a mathematical description for the properties of protein structures, for their correlation, and for their significance.

Computer Simulation↗
PubMed1992

Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.

Apoenzymes↗
PubMed1992

Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction.

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.

Alanine↗
PubMed1992

Quantitative analysis of protein far UV circular dichroism spectra by neural networks.

A new method based on neural network theory is presented to analyze and quantify the information content of far UV circular dichroism spectra. Using a backpropagation network model with a single hidden layer between input and output, it was possible to deduce five different secondary structure fractions (helix, parallel and antiparallel beta-sheet, beta-turn and random coil) with satisfactory correlations between calculated and measured secondary structure data. We demonstrate that for each wavelength interval a specific network is suitable. The remaining discrepancy between the secondary structure data from neural network prediction and crystallography may be attributed to errors in the determination of protein concentration and random noise in the CD signal, as indicated by simulations.

Circular Dichroism↗