Biomedical subjects

M Rheinnecker

Publications and source records attributed to M Rheinnecker.

8 recordsLinked to original sources

PubMed1996

Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

Amino Acid Sequence↗

PubMed1995

Structural factors contributing to the hydrophobic effect: the partly exposed hydrophobic minicore in chymotrypsin inhibitor 2.

The structural basis for the stability of a partly solvent-exposed hydrophobic minicore, formed by the residues Leu51, Val57, and Phe69 in the reactive loop of the serine protease inhibitor chymotrypsin inhibitor 2 (CI2), was analyzed from the stability of 17 mutant proteins, in which side chain methylene groups were deleted or rearranged. The mutations destabilize the protein by 0.3-4.8 kcal/mol, an average of 0.6 kcal per removed methylene group. Double mutant cycles show significant interaction between individual pairs of side chains. There is an excellent linear correlation (r = 0.995) between the free energy of unfolding relative to wild type (delta delta GU-F) and the packing density nC (the number of methyl and methylene groups within 6 A of the removed atoms). delta delta GU-F correlates more weakly with changes in solvent-accessible surface area upon mutation. The correlation improves when the change in solvent-accessible surface are upon mutation is separated into distinct contributions from polar atoms that are able to hydrogen bond to solvent (delta AHB) and from nonpolar atoms (delta AHP). There is also a correlation, however, between packing density and changes in surface area. Elsewhere in CI2, delta delta GU-F for mutations in the buried hydrophobic core correlates best with packing density, whereas in the exposed surface of the alpha-helix, the best correlation is with change in surface area.(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsin↗

PubMed1994

Variants of subtilisin BPN' with altered specificity profiles.

A strategy for increasing the size of the S4 binding pocket was used to improve the specificity of subtilisin BPN' toward substrates with large hydrophobic P4 side chains. This approach involves single and double amino acid replacements at positions 104, 107, and 126. Previously, alteration of I107 to glycine has been found to increase the specificity of subtilisin toward leucine, isoleucine, and phenylalanine as P4 residues by up to 214-fold. Replacement of Y104 by alanine also yields a similar improvement in specificity. However, this subtilisin variant favors isoleucine and phenylalanine over leucine. When L126 was replaced by valine, alanine, and glycine, respectively, only the L126A subtilisin variant, which possesses a 28-fold-increased catalytic efficiency for isoleucine compared with all other substrates tested, showed a significantly improved specificity profile. As inferred from the double-mutant enzymes I107G/L126V, I107G/L126A, and I107G/Y104A, none of the effects of the single amino acid replacements on the kinetic parameters are additive. The I107G/L126V mutant subtilisin has the largest improvement in P4 substrate specificity reported so far: kcat/KM is increased 340-fold for leucine compared to alanine. By contrast, the specificity profile of the I107G/Y104A mutant enzyme is impaired in comparison with that of the corresponding single mutants. Therefore, the design of high-specificity subtilisin variants through the combination of single amino acid replacements in the S4 pocket appears to be nontrivial due to the interference of the introduced structural changes.

Amino Acid Sequence↗

PubMed1993

Hydrolysis of small peptide substrates parallels binding of chymotrypsin inhibitor 2 for mutants of subtilisin BPN'.

Variants of subtilisin BPN' that possess improved specificity towards isoleucine compared with alanine at the P4 position of small peptide substrates, were analysed for their ability to bind chymotrypsin inhibitor 2. The binding of the inhibitor with isoleucine (wild-type) and with alanine as the P4 residue parallels the hydrolysis of tetrapeptide substrates. There is a linear relationship between the free energy of binding of the transition state of the substrate and the free energy of binding of the inhibitor with a slope of 2.0. The data suggest that the inhibitor uses predominantly ground state rather than transition state binding energy.

Amino Acid Sequence↗

PubMed1993

Folding of subtilisin BPN': role of the pro-sequence.

Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate. This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence. The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state. Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence. The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously. However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule. The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed. Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding. Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7.5 kcal/mol; folding is accelerated more than five orders of magnitude.

Amino Acid Sequence↗

PubMed1993

Engineering a novel specificity in subtilisin BPN'.

The specificity of subtilisin BPN' toward substrates with large hydrophobic P4 residues has been improved by single amino acid replacements at positions 104 and 107. Mutations were designed to (i) increase the size of the P4 binding pocket by replacing Ile107, which is at the bottom of the S4 pocket, by Val, Ala, and Gly and (ii) lose the hydrogen bond between Tyr104 and Ser130 at the entrance of the P4 binding pocket by changing Tyr104 to Phe and thus reduce interactions between small P4 side chains and residue 104. All mutant subtilisins, except for I107V, have increased specificity for residues with large side chains at P4 compared with wild type. Using the conventional definition of specificity as the competition of different substrates for the same enzyme, the I107G mutant subtilisin has one of the largest improvements in substrate specificity reported for subtilisin so far; kcat/KM is increased > 200-fold for Phe compared with Ala as the P4 residue. Further, the activity of I107G toward its specific substrate is comparable to that of the wild-type enzyme. Surprisingly, much of the increase in specificity on mutation of Ile107-->Gly appears to result from a lesion that is transmitted through the structure and effects catalysis. The value of kcat/KM for the small substrate acetyltyrosine ethyl ester, which binds to the S1 pocket, drops by 93% on mutation of Ile107-->Gly. The lesion in subtilisin I107G is complemented, however, on binding of longer substrates that have a large hydrophobic P4 amino acid side chain that can bind in the S4 pocket.

Amino Acid Sequence↗

PubMed1993

Folding of subtilisin BPN': characterization of a folding intermediate.

Subtilisin BPN', an extracellular serine protease from Bacillus amyloliquefaciens, requires a 77 amino acid pro-sequence for correct folding in vivo. We report the observation of a metastable folding intermediate during the refolding of wild-type and a proteolytically inactive mutant subtilisin BPN' that lack the pro-sequence. The addition of the pro-sequence as a separate polypeptide chain results in the folding of the intermediate to the native state. The intermediate state of subtilisin is stable at different temperatures, pH values, and salt concentrations for more than a week and retains its competence for folding. The intermediate state possesses a compactness between that of the native and unfolded states. Although it has native-like secondary structure, it shows no distinct near-UV CD spectrum and has a strongly reduced dispersion in the amide and methyl regions of the 1H NMR spectrum. These indicate considerably less tertiary structure than possessed by the native state. However, the intermediate conformation has regions of stable tertiary structure: it has a high-affinity calcium binding site and, after a first noncooperative transition, unfolds with guanidinium chloride in a cooperative process. These results support a folding mechanism for subtilisin BPN' that comprises a high energy transition state, which is lowered by the interaction with the pro-sequence. The similarity to the folding mechanism of alpha-lytic protease supports the hypothesis that a common folding mechanism has been developed through convergent evolution.

Amino Acid Sequence↗

PubMed1991

A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase.

A temperature-sensitive mutant of Saccharomyces cerevisiae was identified which at the restrictive temperature of 37 degrees C is unable to secrete a number of cell wall-associated proteins and thus resembles previously reported sec mutants. In contrast to other sec mutants, however, both the temperature-sensitive growth and the secretion defects can be repaired by the addition of D-mannose to growth media. We show that the mutant possesses a single, apparently recessive mutation which leads to the production of a thermolabile phosphomannose isomerase.

Hot Temperature↗