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A L Fink

Publications and source records attributed to A L Fink.

At least 37 records · Page 2Linked to original sources
PubMed2000

Export and folding of signal-sequenceless Bacillus licheniformis beta-lactamase in Escherichia coli.

Two genetically engineered variants of the Bacillus licheniformis beta-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DeltaG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli beta-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.

Amino Acid Sequence↗
PubMed2000

An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments.

Synthetic amyloid beta-protein (A beta) is used widely to study fibril formation and the physiologic effects of low molecular weight and fibrillar forms of the peptide on cells in culture or in experimental animals. Not infrequently, conflicting results have arisen in these studies, in part due to variation in the starting conformation and assembly state of A beta. To avoid these problems, we sought a simple, reliable means of preparing A beta for experimental use. We found that solvation of synthetic peptide with sodium hydroxide (A beta x NaOH), followed by lyophilization, produced stocks with superior solubility and fibrillogenesis characteristics. Solubilization of the pretreated material with neutral buffers resulted in a pH transition from approximately 10.5 to neutral, avoiding the isoelectric point of A beta (pI approximately 5.5), at which A beta precipitation and aggregation propensity are maximal. Relative to trifluoroacetate (A beta x TFA) or hydrochloric acid (A beta x HCl) salts of A beta, yields of "low molecular weight A beta" (monomers and/or dimers) were improved significantly by NaOH pretreatment. Time-dependent changes in circular dichroism spectra and Congo red dye-binding showed that A beta x NaOH formed fibrils more readily than did the other A beta preparations and that these fibrils were equally neurotoxic. NaOH pretreatment thus offers advantages for the preparation of A beta for biophysical and physiologic studies.

Amino Acid Sequence↗
PubMed1999

Transient dimer in the refolding kinetics of cytochrome c characterized by small-angle X-ray scattering.

The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule.

Animals↗
PubMed1999

Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH.

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.

Circular Dichroism↗
PubMed1999

Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy.

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.

Amyloid↗
PubMed1999

Association of partially-folded intermediates of staphylococcal nuclease induces structure and stability.

Staphylococcal nuclease forms three different partially-folded intermediates at low pH in the presence of low to moderate concentration of anions, differing in the amount of secondary structure, globularity, stability, and compactness. Although these intermediates are monomeric at low protein concentration (< or =0.25 mg/mL), increasing concentrations of protein result in the formation of dimers and soluble oligomers, ultimately leading to larger insoluble aggregates. Unexpectedly, increasing protein concentration not only led to association, but also to increased structure of the intermediates. The secondary structure, stability, and globularity of the two less-ordered partially-folded intermediates (A1 and A2) were substantially increased upon association, suggesting that aggregation induces structure. An excellent correlation was found between degree of association and amount of structure measured by different techniques, including circular dichroism, fluorescence, Fourier transform infrared spectroscopy (FTIR), and small-angle X-ray scattering. The associated states were also substantially more stable toward urea denaturation than the monomeric forms. A mechanism is proposed, in which the observed association of monomeric intermediates involves intermolecular interactions which correspond to those found intramolecularly in normal folding to the native state.

Chromatography, Gel↗
PubMed1999

Chaperone-mediated protein folding.

The folding of most newly synthesized proteins in the cell requires the interaction of a variety of protein cofactors known as molecular chaperones. These molecules recognize and bind to nascent polypeptide chains and partially folded intermediates of proteins, preventing their aggregation and misfolding. There are several families of chaperones; those most involved in protein folding are the 40-kDa heat shock protein (HSP40; DnaJ), 60-kDa heat shock protein (HSP60; GroEL), and 70-kDa heat shock protein (HSP70; DnaK) families. The availability of high-resolution structures has facilitated a more detailed understanding of the complex chaperone machinery and mechanisms, including the ATP-dependent reaction cycles of the GroEL and HSP70 chaperones. For both of these chaperones, the binding of ATP triggers a critical conformational change leading to release of the bound substrate protein. Whereas the main role of the HSP70/HSP40 chaperone system is to minimize aggregation of newly synthesized proteins, the HSP60 chaperones also facilitate the actual folding process by providing a secluded environment for individual folding molecules and may also promote the unfolding and refolding of misfolded intermediates.

Chaperonins↗
PubMed1998

Conformational changes of an Hsp70 molecular chaperone induced by nucleotides, polypeptides, and N-ethylmaleimide.

Hsp70 molecular chaperones are highly conserved ATPases that guide the folding and assembly of proteins in many cellular pathways. They use the energy of ATP binding and hydrolysis to regulate their interactions with hydrophobic regions of unfolded proteins. The activities and the conformations of the N-terminal nucleotide- and C-terminal polypeptide-binding domains of Hsp70s are coupled. We recently reported that the sulfhydryl-modifying reagent N-ethylmaleimide (NEM) inactivates the yeast Hsp70 Ssa1p by reacting with its three cysteine residues which are located in the nucleotide-binding domain. To further characterize conformational changes associated with interdomain coupling and to determine whether NEM alters Ssa1p's conformation, the structures of Ssa1p and NEM-modified Ssa1p (NEM-Ssa1p) were compared using a variety of biophysical techniques. Size exclusion chromatography revealed that NEM-Ssa1p is more oligomeric and more resistant to nucleotide- or polypeptide-dependent depolymerization than Ssa1p. Measurement of the thermal stability indicated that NEM modification has an effect very similar to that of binding of nucleotides to the unmodified protein. Circular dichroism demonstrated small differences in the secondary structure of Ssa1p and NEM-Ssa1p, and in their complexes with nucleotides. NEM modification increased the ANS fluorescence of Ssa1p and exposed numerous trypsin-sensitive sites in its nucleotide-binding domain. The intrinsic fluorescence of Ssa1p's only tryptophan residue, which is located in a C-terminal alpha-helical region adjacent to the polypeptide-binding cleft, was quenched in the presence of ATP, but not ADP. NEM modification altered nucleotide-dependent changes in the intrinsic fluorescence of Ssa1p. Together, these results demonstrate that NEM alters the conformation of Ssa1p and disrupts, but does not eliminate, interdomain communication. Furthermore, the results provide evidence for a model in which the polypeptide-binding cleft of Hsp70s is covered by an alpha-helical lid that is open in the presence of ATP, but closed in the presence of ADP.

Adenine Nucleotides↗
PubMed1998

Protein denaturation: a small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c.

Solution X-ray scattering was used to study the equilibrium unfolding of cytochrome c as a function of guanidine hydrochloride concentration at neutral pH. The radius of gyration (Rg) shows a cooperative transition with increasing denaturant with a similar Cm to that observed with circular dichroism. However, the lack of an isoscattering point in the X-ray scattering patterns suggests the equilibrium unfolding is not simply a two-state process. Singular value decomposition (SVD) analysis was applied to the scattering patterns to determine the number of distinct scattering species. SVD analysis reveals the existence of three components, suggesting that at least three equilibrium states of the protein exist. A model was employed to determine the thermodynamic parameters and the scattering profiles of the three equilibrium states. These scattering profiles show that one state is native (N). The other two states (U1, U2) are unfolded, with U2 being fully unfolded and U1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U1 is maximally populated at intermediate denaturant concentrations while U2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U1 and U2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.

Animals↗
PubMed1998

Anion-induced folding of Staphylococcal nuclease: characterization of multiple equilibrium partially folded intermediates.

The refolding of acid-unfolded staphylococcal nuclease (SNase) induced by anions was characterized, and revealed the existence of three different partially folded intermediates (A states). The three intermediates lack the rigid tertiary structure characteristic of native states, and differ in their degree of folding as measured by probes of secondary structure, size, stability and globularity. The least structured conformation, A1, is stabilized by chloride (600 mM) or sulfate (100 mM). It is about 50% folded (based on circular dichroism and small angle X-ray scattering (SAXS) data). The next most structured intermediate, A2, is induced by trifluoroacetate (300 mM) and has approximately 70% native-like secondary structure. The most structured intermediate, A3, is stabilized by trichloroacetate (50 mM) and has native-like secondary structure content and is almost as compact as the native state. The stability toward urea denaturation increases with increasing structure of the intermediates. Moreover, ureainduced unfolding studies show that these partially folded species are separated from each other, and from the unfolded state, by significant free energy barriers, suggesting that they are distinct conformational states. Kratky plots, based on the SAXS data, indicate that the two more structured intermediates have significant globularity (i.e. a tightly packed core), whereas the less structured intermediate has very little globularity. These observations support a model of protein folding in which certain conformations are of particularly low free energy and hence populated under conditions which differentially destabilize the native state. These partially folded intermediates probably consist of ensembles of substates with a common core of native-like secondary structure, which is responsible for their stability. Consequently, it is likely that the intermediates observed here represent the equilibrium counterparts of transient kinetic intermediates.

Anions↗
PubMed1998

Association-induced folding of globular proteins.

It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.

Circular Dichroism↗
PubMed1998

Time-resolved circular dichroism studies of protein folding intermediates of cytochrome c.

The circular dichroism spectra of cytochrome c (cytc) in 4.6 M guanidine hydrochloride (pH 6.5) indicate that the secondary structure in reduced cytc is near-native, whereas in the CO-bound species (COCytc) it is substantially unfolded. Photolysis of COCytc should thus induce large changes in the secondary structure, which can be probed with time-resolved circular dichroism (TRCD) spectroscopy in the far-UV region. Time-resolved absorption (TROA) and TRCD methods were used to study the photolysis reaction of COCytc in efforts to identify structural intermediates in cytc folding on time scales from nanoseconds to seconds. TROA data from the Soret region, similar to previous studies, showed four intermediates with lifetimes of 2, 50, 225, and 880 micros. The 2-micros process is proposed to involve Fe(II)-Met80 coordination. Approximately 7% of the native CD signal was observed in the TRCD signal at 220 nm within 500 ns, with no significant additional secondary structure formation observed. Further folding after 2 micros may be inhibited by ligation of His26/His33 with Fe(II), which is suggested to be associated with the 50-micros phase. The two slowest components, tau = 225 and 880 micros, are attributed to CO rebinding on the basis of mixed-gas experiments. CO rebinding is expected to compete with protein folding and favor the unfolded state. However, when the two CO rebinding lifetimes are extended into milliseconds by reducing the CO concentration, there is still no significant increase in CD signal at 220 nm.

Animals↗
PubMed1998

A new attenuated total reflectance Fourier transform infrared spectroscopy method for the study of proteins in solution.

An attenuated total reflectance Fourier transform infrared method has been developed that allows collection of spectra from proteins in solution. This method eliminates any structural perturbations induced by the internal reflection element (IRE), and thus the spectra reflect the solution conformation of the protein. A key feature of the method is subtraction of the signal from any protein adsorbed to the IRE. The advantages of this method include the small amount of sample required and the high sampling rate. Attenuated total reflectance (ATR)-Fourier transform infrared spectroscopy (FTIR) is more versatile than transmission FTIR because it is possible to collect spectra of nontransparent samples, to use samples of very low protein concentration (< or = 0.3 mg/ml), and to study proteins in the presence of strongly absorbing solutes (such as denaturants). The experimental procedures and data processing routines developed were evaluated by collecting spectra from a set of 13 proteins and evaluating their accuracy with a partial least-squares analysis. The relative mean and standard deviation errors for the basis set analysis were 6.3% for alpha-helix, 5.9% for beta-sheet/extended structure, and 4.4% for turn, which are similar to values from comparable analyses of transmission FTIR spectra. In addition, a detailed comparison between this solution ATR method and the hydrated thin-film ATR technique is presented.

Adsorption↗
PubMed1998

Protein aggregation: folding aggregates, inclusion bodies and amyloid.

Aggregation results in the formation of inclusion bodies, amyloid fibrils and folding aggregates. Substantial data support the hypothesis that partially folded intermediates are key precursors to aggregates, that aggregation involves specific intermolecular interactions and that most aggregates involve beta sheets.

Amyloid↗
PubMed1998

Discrete intermediates versus molten globule models for protein folding: characterization of partially folded intermediates of apomyoglobin.

BACKGROUND: Although small proteins may fold in an apparent two-state manner, most studies of protein folding reveal transient intermediates. The 'molten globule' has been proposed to be a general intermediate in protein folding. Relatively little is known about the structure of partially folded intermediates, however. RESULTS: Three different partially folded intermediates of apomyoglobin, having 35%, 50% and 60% helix, were characterized at low pH in the presence of different anions. It was found that increasing helical structure correlated with decreasing size and increasing stability to urea. Similar intermediates have been observed transiently during the folding of apomyoglobin. CONCLUSIONS: The results are consistent with a model for folding in which structural units coalesce to form a core of relatively native-like structure, the remainder of the protein being relatively disordered. For a given protein there will be certain partially folded conformations of particularly low free energy that are preferentially populated under both equilibrium and transient folding conditions. The conformation and topology of the intermediates will be specific to a given protein, so there are no 'general' intermediates, such as the molten globule, in folding.

Animals↗
PubMed1998

Structural effect of association on protein molecules in partially folded intermediates.

Fluorescence and circular dichroism spectroscopy, small angle X-ray scattering and high performance liquid gel filtration have shown that oligomerization considerably affects the structural properties and conformational stability of partially folded intermediates of staphylococcal nuclease. Conformational transitions induced by different anions and association in the acid-unfolded protein are described. It is shown that association of non-native conformations of the protein molecule can be an additional structuring factor. The corresponding folding schemes and phase diagrams are suggested.

Dimerization↗
PubMed1998

Structural properties of staphylococcal nuclease in oligomeric A-forms.

Association affects the structural properties of different partially folded conformations of staphylococcal nuclease induced by anions of different nature. It is shown that oligomerization induces new structural levels in non-native A-forms. A close structural similarity between the monomeric A2 and the dimeric (A1)2 forms as well as between the monomeric A3 and oligomeric [(A1)2]M and [A2]M forms is established. This suggests that association of a protein molecule in partially folded conformations can be an additional structure forming factor.

Anions↗