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A L Fink

Publications and source records attributed to A L Fink.

At least 55 records · Page 3Linked to original sources
PubMed1997

Effect of prolyl isomerase on the folding reactions of staphylococcal nuclease.

The low-temperature fluorescence-detected refolding of staphylococcal nuclease (SNase) can be described by three slow kinetic phases. The slowest phase is absent in the P117G mutant of SNase. Peptidyl prolyl cis-trans isomerase (cyclophilin), which has been shown to catalyze the slow folding reactions of some proteins, was employed to determine which of the refolding reactions of SNase and P117G SNase involve proline isomerization. We report here that all three folding phases of the wild type and the slower phase of P117G SNase are catalyzed by prolyl isomerase, indicating that proline isomerization is involved in these fluorescence-detected phases in the refolding of SNase. Since the rates of these phases are denaturant-dependent, we conclude that the slow folding steps involve isomerization of non-native cis proline peptide bonds and are tightly coupled to denaturant-sensitive structural changes.

Catalysis↗
PubMed1997

A point mutation leads to altered product specificity in beta-lactamase catalysis.

beta-Lactamases are the primary cause of beta-lactam antibiotic resistance in many pathogenic organisms. The beta-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A beta-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L beta-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of beta-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.

Asparagine↗
PubMed1997

The penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis is embedded in the plasma membrane via a four-alpha-helix bundle.

Prediction studies, conformational analyses and membrane-topology mapping lead to the conclusion that the penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis, is embedded in the plasma membrane bilayer via four transmembrane segments TM1-TM4 that form a four-alpha-helix bundle. The extracellular 262-amino-acid-residue polypeptide, S340-R601, that is fused at the carboxy end of TM4, possesses the amino acid sequence signature of a penicilloyl serine transferase. It probably functions as penicillin sensor. As an independent entity, this polypeptide behaves as a high-affinity penicillin-binding protein. As a component of the full-size BlaR, it adopts a different conformation presumably because of interactions with the extracellular 63-amino-acid-residue P53-S115 loop that connects TM2 and TM3. Reception of the penicillin-induced signal requires a precise conformation of the sensor but it does not involve penicilloylation of the serine residue S402 of motif STYK. Signal transmission through the plasma membrane by the four-alpha-helix bundle may proceed in a way comparable to that of the aspartate receptor, Tar. Signal emission in the cytosol by the intracellular 189-amino-acid-residue Y134-K322 loop that connects TM3 and TM4, may proceed via the activation of a putative metallopeptidase.

Amino Acid Sequence↗
PubMed1996

Conformational characterization of DnaK and its complexes by small-angle X-ray scattering.

DnaK, a member of the 70 kDa heat shock protein (hsp70) family, and its complexes with substrate proteins and nucleotides were characterized by small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC) techniques. The SAXS data indicated that DnaK has a dumbbell-shaped structure with a maximum dimension (dmax) of 112 angstrom, which is consistent with the reported two major functional domains [Chappell et al. (1987) J. Biol. Chem. 268, 12730-12735; Flaherty et al. (1990) Nature 346, 623-628]. The data were best fit by a model in which the two domains either are connected by a short hinge region or are just in contact with each other. The radius of gyration (Rg) of DnaK was determined as 37.5 +/- 1.0 angstrom in the absence of nucleotide. Binding of ATP induces a conformational change in DnaK as reflected by the changes in its P(r) function and Kratky plot, the increases (1-2 angstrom) in both its radius of gyration (Rg) and its Stokes radius (Rs), and the increase in its dmax (5-10 angstrom ). SAXS and SEC-HPLC results indicate that the association state of DnaK is very sensitive to the buffer concentration and the presence of substrates, as well as the protein concentration. At high buffer and protein concentrations, DnaK dimerizes, resulting in an increase in its apparent Rg and dmax values. The addition of substrate (unfolded protein or ATP) results in a return to the Rg value of monomeric DnaK, due to the dissociation of DnaK multimers induced by the substrate binding and resultant conformational changes. The DnaK-substrate protein complex gives a smaller Rg than expected, suggesting that the substrate protein binds to a cavity or cleft on DnaK rather than the exterior of the chaperone. The Kratky plot of the Gdn.HCl-induced unfolding intermediate state of DnaK is consistent with a compact, molten globule-like conformation, as previously suggested based on CD, fluorescence, and SEC-HPLC results [Palleros et al. (I 993) Biochemistry 32, 4314-4321].

Bacterial Proteins↗
PubMed1996

Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex.

Many of the functions of the Escherichia coli Hsp70, Dnak, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a Kd estimated at < or = 50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (kan > 1 s-1), whereas the off-rate is very slow on the HPLC timescale (kant < or = 10(-4) s-1). GrpE has no affinity for ATP or ADP, nor the oligomeric and molten globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary structural change in DnaK to that induced by binding of ATP, based on the blue shift in lambda max from the fluorescence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.

Adenosine Diphosphate↗
PubMed1995

Substitution of Asp for Asn at position 132 in the active site of TEM beta-lactamase. Activity toward different substrates and effects of neighboring residues.

Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM beta-lactamase, and selective screening in ampicillin-plates, we obtained the N132D mutant enzyme. The kinetic characterization of this mutant indicated relatively small effects compared to the wild-type. Both pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK's for the wild type. This effect was predominantly due to changes in Km. In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km for several side chain-containing penicillin substrates increases when the pH is above 5.5. 6-Aminopenicillanic acid, which lacks a side chain, did not show this effect. With benzylpenicillin, ampicillin, and carbenicillin, kcat for the mutant showed a similar pH dependence as the wild type. With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type. The nature of the 104 side chain may affect the environment of Asp132; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to bacterial cells. The computed contact interactions from modeling substrate complexes between benzylpenicillin or 6-aminopenicillanic acid with the N132D mutant confirmed the importance of the protonation state of residue Asp132 for the complex stability with side chain-containing substrates. The data indicate that the contact between the side chain of residue 132 and the substrate is relevant for the ground state recognition, but because of close contact with several important groups in its neighborhood, residue 132 is also indirectly involved in the catalytic step of the wild-type enzyme.

Asparagine↗
PubMed1995

A new TEM beta-lactamase double mutant with broadened specificity reveals substrate-dependent functional interactions.

Using a random combinatorial mutagenesis of TEM beta-lactamase, directed against residues potentially involved in substrate discrimination, followed by selection on third generation cephalosporins, we obtained the double mutant E104M/G238S. Additionally, by using cloning strategies and site-directed mutagenesis we constructed the individual single mutants and also the single modification E104K and the double mutant E104K/G238S, which broaden the specificity of clinically isolated TEM beta-lactamase variants. The kinetic characterization of the purified double mutant E104M/G238S and its single counterparts E104M and G238S was carried out. The single mutant E104M exhibited increased kcat values against all substrates tested. Km values remained similar to the values shown by the wild-type enzyme. The mutation at E104M was responsible for the increased hydrolysis rate against cefuroxime shown by the double mutant E104/G238S. The effect of mutation G238S varied more pronouncedly, depending on the substrate. In general, a lower Km was observed, but also a decreased kcat. The double mutant E104M/G238S exhibited a higher hydrolytic rate against cefotaxime compared with the corresponding single mutations. We observed nearly a 1000-fold greater kcat/Km for the double mutant than for the wild type. This improvement in catalysis was the consequence of increased kcat and decreased Km values. Computed contact interactions from modeling substrate complexes show reliable results only for benzylpenicillin. The modeling results with this substrate confirmed the observed enzyme activities for the different single and double mutants. Analysis of the apparent coupling energies, as calculated from the kinetic parameters of the single and double mutants, showed that the quantitative effect of a second mutation on a single mutant was either absent, additive, partially additive, or synergistic with respect to the first mutation, depending on the substrate analyzed.

Drug Resistance, Microbial↗
PubMed1995

Compact intermediate states in protein folding.

The number of observations of both stable and transient partially folded intermediates is now sufficient for us to assume that all proteins, under the appropriate conditions, will form such species. These intermediates are characterized by substantial secondary structure and little tertiary structure; a collapsed conformation more compact than the unfolded state; exposed hydrophobic surfaces, which lead to binding of hydrophobic dyes and a propensity to aggregate; and a heat capacity similar to that of the unfolded state. The term compact intermediates encompasses a broad range of conformations and degrees of folding and compactness: compact intermediates have no single, unique conformation, but rather a whole plethora of structures that range from being very similar to the native state to being substantially expanded and significantly unfolded. The properties of compact intermediates from different proteins, and in some cases from the same protein under different conditions, may be significantly different. Equilibrium compact intermediates may be good models for transient intermediates formed during folding.

Models, Chemical↗
PubMed1995

Molten globules.

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Models, Chemical↗
PubMed1994

Classification of acid denaturation of proteins: intermediates and unfolded states.

A systematic investigation of the effect of acid on the denaturation of some 20 monomeric proteins indicates that several different types of conformational behavior occur, depending on the protein, the acid, the presence of salts or denaturant, and the temperature. Three major types of effects were observed. Type I proteins, when titrated with HCl in the absence of salts, show two transitions, initially unfolding in the vicinity of pH 3-4 and then refolding to a molten globule-like conformation, the A state, at lower pH. Two variations in this behavior were noted: some type I proteins, when titrated with HCl in the absence of salts, show only partial unfolding at pH 2 before the transition to the molten globule state; others of this class form an A state that is a less compact from of the molten globule state. In the presence of salts, these proteins transform directly from the native state to the molten globule conformation. Type II proteins, upon acid titration, do not fully unfold but directly transform to the molten globule state, typically in the vicinity of pH 3. Type III proteins show no significant unfolding to pH as low as 1, but may be caused to behave similarly to type I in the presence of urea. Thus, the exact behavior of a given protein at low pH is a complex interplay between a variety of stabilizing and destabilizing forces, some of which are very sensitive to the environment. In particular, the protein conformation is quite sensitive to salts (anions) that affect the electrostatic interactions, denaturants, and temperature, which cause additional global destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗
PubMed1994

Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase.

Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site. Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis. This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties. In particular, in comparison to the wild-type, kcat. for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates. For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme. Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation. Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity. We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding.

Bacillus↗
PubMed1994

Protein conformational changes induced by 1,1'-bis(4-anilino-5-naphthalenesulfonic acid): preferential binding to the molten globule of DnaK.

1,1'-Bis(4-anilino-5-naphthalenesulfonic acid) (bis-ANS), a hydrophobic fluorescent molecular probe which has been shown to bind to compact intermediate states of proteins (molten globules) and also to many nucleotide binding sites, induces a conformational change in DnaK by preferentially binding to its partially folded intermediate state (I) and thus shifting the equilibrium from favoring the native state (N) to favoring the I state. The conformational change was detected by CD, fluorescence emission, size exclusion chromatography, and small-angle X-ray scattering. The presence of bis-ANS significantly decreases the midpoint, Tm, of the initial transition (N-->I) in the thermal unfolding of DnaK, resulting in the apparent destabilization of the native state of DnaK. There is a linear correlation between the apparent free energy (reflected by Tm) of this transition and the concentration of bis-ANS. Bis-ANS does not affect the midpoint of the transition for DnaK from the intermediate to the unfolded state (U). An additional small transition from I to I*, a more expanded intermediate state, was observed, suggesting that the thermal denaturation of DnaK proceeds via a four-state (N-->I-->I*-->U) unfolding process. The addition of nucleotides, ADP or ATP, to the DnaK-bis-ANS complex causes a decrease in bis-ANS fluorescence emission due to the release of bound bis-ANS from the intermediate state of DnaK. This is due to preferential binding of the nucleotide to the native state of DnaK, resulting in a shift in the equilibrium from the intermediate toward the native state rather than the direct displacement of bis-ANS bound in the nucleotide binding site. Denaturation of DnaK induced by bis-ANS can be minimized by working at a temperature much lower than the Tm of the protein, at low dye concentration, and in the presence of nucleotide. Under these conditions, bis-ANS binds to the native state of DnaK.

Adenosine Diphosphate↗
PubMed1994

Site-directed mutagenesis of glutamate-166 in beta-lactamase leads to a branched path mechanism.

Glutamate-166 of the Bacillus licheniformis beta-lactamase was specifically mutated to aspartate and cysteine in order to probe the function of this residue in catalysis. In both cases, a large decrease in activity (kcat/Km was 3.5 x 10(-5) smaller for E166C and 1 x 10(-3) smaller for E166D than for the wild-type) was observed, although the kinetics for the two mutants were very different. The pH-rate profiles for E166D and E166C reflected the ionization characteristics of the new residue at site 166. This result indicates that the ionization of Glu-166 is responsible for the acidic limb of the kcat/Km-pH profiles, and suggests that the function of Glu-166 is that of a general base catalyst. The kinetics of the E166C mutant were investigated in detail. An initial burst was observed, whose amplitude was stoichiometric with the enzyme concentration, suggesting rate-limiting deacylation of the acyl-enzyme intermediate. However, further study revealed that in the presence of 0.5 M sodium sulfate, which stabilizes the native conformational state, the magnitude of the burst corresponded to 2 equiv of enzyme. This observation, in conjunction with the limited effect of the mutation on Km, indicated that the mutation resulted in a change in the kinetic mechanism from the linear, acyl-enzyme pathway to one with a branch leading to an inactive form of the acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1994

hsp70-protein complexes. Complex stability and conformation of bound substrate protein.

The presence of bound substrate protein increases the thermostability of hsp70 molecular chaperones (heat shock proteins of molecular mass 70 kDa). Complexes between hsp70 and unfolded substrate proteins were isolated by size-exclusion high performance liquid chromatography. The isolated complexes were observed to dissociate at a significant rate even in the absence of ATP. The presence of ADP caused a substantial increase in the stability of the complex. Both ADP and inorganic phosphate were found to inhibit the ATP-induced dissociation of complex. ADP was also observed to increase both the rate of complex formation and its stability as a function of temperature, suggesting an important regulatory role for nucleotides during heat shock. Circular dichroism and fluorescence studies of the complex between DnaK and a thermally unstable mutant of staphylococcal nuclease indicate that the bound substrate protein is significantly unfolded. A model for hsp70 cycle of complex formation and dissociation, which accounts for the regulatory role of nucleotides, is proposed.

Adenosine Diphosphate↗
PubMed1994

Nativelike secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR.

Attenuated total reflectance FTIR has been used to study the structure of human interleukin-1 beta in inclusion bodies (IBs) and other aggregated forms. The secondary structure composition of native wild-type IL-1 beta determined by FTIR is in excellent agreement with that previously determined by crystallography and NMR: 52% beta-sheet, 25% loop/irregular structure, and 23% turn. Remarkably, IL-1 beta inclusion bodies exhibit secondary structural composition very similar to that of the native protein. The results indicate that the IBs form from a folding intermediate that has nativelike secondary structure. The secondary structure content of aggregated IL-1 beta, formed either in refolding or by thermal denaturation, was identical within experimental error to that of the IB, indicating that these aggregates were formed from intermediates with structures similar to that of the inclusion body.

Aged↗
PubMed1994

Thermally denatured ribonuclease A retains secondary structure as shown by FTIR.

Fourier transform-infrared (FTIR) spectroscopy has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A) at pH* 2.0 (uncorrected pH measured in D2O). The amide I spectral region of the native and thermally denatured protein was compared. A substantial decrease in the amount of beta-sheet and alpha-helix and a corresponding increase in the amount of turn and unordered structure was observed on thermal denaturation. The results indicate that thermally denatured RNase A contains significant amounts of secondary structure (11% helix and 17% beta-sheet), consistent with previous results reported for circular dichroism, and with a relatively compact structure, as revealed by dynamic light scattering. These results are in contrast to those of amide protection experiments reported recently [Robertson, A.D., & Baldwin, R.L. (1991) Biochemistry 30, 9907-9914] which indicated no stable hydrogen-bonded structure under these experimental conditions. Possible explanations for this apparent discrepancy are given.

Animals↗