Characterization of MB-1. A dimeric helical protein with a compact core.
(73/6430)
MB-1 is a de-novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein. MB-1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases [Beauregard, M., Dupont, C., Teather, R.M. & Hefford, M.A. (1995) Bio/Technology 13, 974]. Here we report an analysis of the secondary, tertiary and quaternary structures in MB-1 using circular dichroism, fluorospectroscopy and size-exclusion chromatography. Our data indicate that the MB-1 structure is close to the target structure, an alpha-helical bundle, in many respects and is highly helical in solution. The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design. Furthermore, MB-1 was found to be native-like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB-1 has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins. On the other hand, our analysis revealed two aspects that command further attention. The folding stability of MB-1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size. Size-exclusion chromatography experiments and analysis of MB-1 denaturation indicate that MB-1 is dimeric, not monomeric as designed. In light of these results, the utility and the current limitations of our design approach are discussed. (+info)
A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants.
(74/6430)
Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm. (+info)
Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.
(75/6430)
An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions. (+info)
Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.
(76/6430)
In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed. (+info)
A stable intermediate in the equilibrium unfolding of Escherichia coli citrate synthase.
(77/6430)
Urea-induced unfolding of Escherichia coli citrate synthase occurs in two phases, as monitored by circular dichroism at 222 nm (measuring secondary structure) or by tryptophan fluorescence. In this paper we characterize the intermediate state, which retains about 40% of the ellipticity of the native state, and is stable between 2.5 M and 5.5 M urea, approximately. This intermediate binds significant amounts of the probe for hydrophobic surfaces, anilinonaphthalene sulfonate, but forms aggregates at least as high as an octamer, as shown by transverse urea gradient polyacrylamide electrophoresis. Thermal denaturation of E. coli citrate synthase also produces an intermediate at temperatures near 60 degrees C, which also retains about 40% of the native ellipticity and forms aggregates, as measured by electrospray-ionization/time-of-flight mass spectrometry. We have used a collection of "cavity-forming" mutant proteins, in which bulky buried hydrophobic residues are replaced by alanines, to explore the nature of the intermediate state further. A certain amount of these mutant proteins shows a destabilized intermediate, as measured by the urea concentration range in which the intermediate is observed. These mutants are found in parts of the citrate synthase sequence that, in a native state, form helices G, M, N, Q, R, and S. From this and other evidence, it is argued that the intermediate state is an aggregated state in which these six helices, or parts of them, remain folded, and that formation of this intermediate is also likely to be a key step in the folding of E. coli citrate synthase. (+info)
Observation of strange kinetics in protein folding.
(78/6430)
Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant. The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state. The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding. (+info)
Stretching single-domain proteins: phase diagram and kinetics of force-induced unfolding.
(79/6430)
Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a stepwise "quantized" manner. Unfolding dynamics and forces required to stretch proteins depend sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value, which is determined by the barrier to unfolding when f = 0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single-molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants. (+info)
Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana. Evidence for cold denaturation in regions outside of the DNA binding domain.
(80/6430)
A transcriptional activator, CBF1, from Arabidopsis thaliana, which has the AP2 domain for DNA binding and regulates the cold acclimation response, was overexpressed in Escherichia coli, purified, and characterized. Analyses of the interaction between CBF1 and the C-repeat/dehydration-responsive element by fluorescence measurement showed that CBF1 binds to C-repeat/dehydration-responsive element as a monomer irrespective of the temperature. CD spectra of the intact and truncated CBF1 proteins (1-213, 41-213, 41-157, and 41-146) were measured to examine the temperature-dependent changes of the secondary structure of CBF1. The results suggested that the CBF1 protein has regions exhibiting reversible cold denaturation in the range between 30 and -5 degrees C and also has a region exhibiting thermal denaturation between 40 and 60 degrees C. This cold denaturation occurred in both the N-terminal and acidic regions. The thermal denaturation occurred in the region encompassing the AP2 domain. The difference between the retention time of CBF1 at 4 degrees C and that at 25 degrees C in gel filtration, and the decrease of the sedimentation coefficient, s20,w, caused by the temperature change from 25 to 3 degrees C, strongly suggested that the cold denaturation was accompanied by the extension of the molecule. The possible cold denaturation observed here might be a physiologically important structural response of CBF1 to cold stress. (+info)