PubMed · 7300475
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J W Snellen, K S Chang. 1981. Calorimeter ergometer for concentric and eccentric work.. https://doi.org/10.1007/bf02442557
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The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis. Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely. Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea. The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event. These results are also clearly incompatible with the early proposals on the structure of the intermediate. It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2. These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.
Gelatin-containing microemulsion based organogels have been used as an immobilisation matrix for lipases from a number of different sources. Kinetic resolutions of octan-3-ol, 1-octen-3-ol and 1-octyn-3-ol by esterification with decanoic acid have been performed using Chromobacterium viscosum (CV) lipase. CV lipase is highly enantioselective in favour of the (R)-(-) isomer of octan-3-ol, but the enantioselectivity is both reversed and decreased by the introduction of unsaturation at the 1-position. Marked improvements in enantioselectivity were achieved by carrying out the reaction at -15 degrees C, the enantiomeric excess of the ester product increasing from 47% (E = 3) to 73% (E = 8) in the case of 1-octen-3-ol, and from 17% (E = 1.4) to 38% (E = 2.5) in the case of 1-octyn-3-ol. The enantiomeric excess was approximately 85% (E approximately 15) for octan-3-ol, and there was no marked improvement in enantioselectivity even at -15 degrees C. Apparent activation energies for the esterification using decanoic acid of octan-3-ol, 1-octen-3-ol and 1-octyn-3-ol by CV lipase were 32 kJ mol-1, 31 kJ mol-1 and 41 kJ mol-1, respectively. This compares to an activation energy of 21 kJ mol-1 for the esterification of octan-1-ol with decanoic acid using CV lipase under the same conditions. Lipases from Pseudomonas (Fluka), Pseudomonas (Genzyme) and lipoprotein lipase ex Microbial (Genzyme) also selectively esterified the (R)-(-) isomer of racemic octan-3-ol, the two Pseudomonas preparations yielding product with an enantiomeric excess of 90%. Candida cylindracea lipase did not exhibit activity in gelatin-containing MBGs. Large-scale syntheses were performed in a 1 dm3 batch reactor in which 200 cm3 of pelleted MBG (containing 350 mg of CV lipase) was used repeatedly for the kinetic resolution of octan-3-ol.
Ribose-binding protein (RBP) has a bilobate structure and functions in the periplasm of Escherichia coli. Mutations that affect the folding of RBP were isolated as intragenic suppressors for the export-defective signal sequence mutation. Of 13 different mutational changes found in the mature region, 12 were located in the several peptides forming the N-domain, and one in the C-domain. Translocation kinetics of mutant proteins were analyzed by pulse-labeling and chase experiments, showing the recovery of precursor processing in the range of 42 to 70%. Folding properties of seven mutant RBPs purified were investigated in vitro by means of tyrosine fluorescence. The stability of the mutant proteins, estimated by equilibrium analysis in the presence of denaturant, were reduced by 2.1 to 5.1 kcal/mol of changes in free energy of unfolding. All the mutant proteins showed retardation in folding rate by 4.4 to 63-fold compared to wild-type while unfolding was little affected. The only exception was the L129Q that has a change in the C-domain resulting in unstability due to faster unfolding. Our approach took advantage of an involvement of the folding process in protein export, which was genetically employed to dissect the folding pathway of RBP. As a result, amino acid residues that are specifically involved in the folding pathway of RBP were identified. Most of them are concentrated in one of the subdomains, suggesting that the folding event in the N-domain of RBP is crucial in the rate-determining step.