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At least 451 records · Page 25Linked to original sources
PubMed1993

Searching sequence space to engineer proteins: exponential ensemble mutagenesis.

We describe an efficient method for generating combinatorial libraries with a high percentage of unique and functional mutants. Combinatorial libraries have been successfully used in the past to express ensembles of mutant proteins in which all possible amino acids are encoded at a few positions in the sequence. However, as more positions are mutagenized the proportion of functional mutants is expected to decrease exponentially. Small groups of residues were randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. By using optimized nucleotide mixtures deduced from the sequences selected from the random libraries, we have simultaneously altered 16 sites in a model pigment binding protein: approximately one percent of the observed mutants were functional. Mathematical formalization and extrapolation of our experimental data suggests that a 10(7)-fold increase in the throughput of functional mutants has been obtained relative to the expected frequency from a random combinatorial library. Exponential ensemble mutagenesis should be advantageous in cases where many residues must be changed simultaneously to achieve a specific engineering goal, as in the combinatorial mutagenesis of phage displayed antibodies. With the enhanced functional mutant frequencies obtained by this method, entire proteins could be mutagenized combinatorially.

Amino Acid Sequence↗
PubMed2004

Sortase-mediated protein ligation: a new method for protein engineering.

Sortase (SrtA), a transpeptidase from Staphylococcus aureus, catalyzes a cell-wall sorting reaction at an LPXTG motif by cleaving between threonine and glycine and subsequently joining the carboxyl group of threonine to an amino group of pentaglycine on the cell wall peptidoglycan. We have applied this transpeptidyl activity of sortase to in vitro protein ligation. We found that in the presence of sortase, protein/peptide with an LPXTG motif can be specifically ligated to an aminoglycine protein/peptide via an amide bond. Additionally, sortase can even conjugate substrates such as (d)-peptides, synthetic branched peptides, and aminoglycine-derivatized small molecules to the C terminus of a recombinant protein. The sortase-mediate protein ligation is robust, specific, and easy to perform, and can be widely applied to specific protein conjugation with polypeptides or molecules of unique biochemical and biophysical properties.

Amino Acid Sequence↗
PubMed2004

Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering.

Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca2+ situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca2+-modulated kinetics of the AMY2/BASI interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors.

Calcium↗
PubMed1991

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells.

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.

Amino Acid Sequence↗
PubMed2000

Understanding peptide interactions with the lipid bilayer: a guide to membrane protein engineering.

In recent years, studies of the interactions of designed hydrophobic and amphipathic polypeptides with biological membranes have progressed considerably. In-plane and transmembrane helical domains have been engineered as well as sequences that exhibit dynamic distributions of different topologies. These sequences not only help our understanding of the thermodynamic interaction contributions that determine peptide orientation, but also exhibit interesting biological functions as autonomous units. In addition, helices are considered to be folding intermediates of multi-spanning membrane proteins. The specificity and thermodynamic stability of transmembrane helix-helix interactions or the assembly of beta sheets at the membrane surface have, therefore, become a focus of ongoing research.

Animals↗
PubMed2002

Protein engineering for crystallization of the GTPase Sar1 that regulates ER vesicle budding.

Sar1 is an important and unique GTPase that regulates vesicle budding from the ER membrane. An effort to crystallize full-length hamster Sar1 was unsuccessful owing to the aggregation of Sar1 in solution as indicated by dynamic light-scattering measurements. It was presumed that a patch of hydrophobic residues in the N-terminal region of Sar1 was responsible for the aggregation. To attempt to improve protein crystallizability, the N-terminal residues of Sar1 were progressively truncated and the solution behavior of the resulting Sar1 variants was monitored by dynamic light scattering. Truncation of the first nine residues from the N-terminus led to a Sar1 variant that is monodisperse in solution. This well behaved Sar1 variant yielded crystals in just a few days that were ultimately refined to diffraction quality.

Animals↗
PubMed1995

Stabilization of a strained protein loop conformation through protein engineering.

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.

Crystallography, X-Ray↗
PubMed1993

A type I collagen reporter gene construct for protein engineering studies. Functional equivalence of transfected reporter COL1A1 and endogenous gene products during biosynthesis and in vitro extracellular matrix accumulation.

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by site-directed mutagenesis of the mouse COL1A1 gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino acid 822 of the triple helix from methionine to isoleucine. This change allowed the reporter protein, [Ile822]alpha 1(I), to be distinguished from the wild-type alpha 1(I), and quantified, by its altered CNBr cleavage pattern. In mouse Mov13 cells, which synthesize no endogenous pro alpha 1(I), reporter chains associated with endogenous pro alpha 2(I), formed pepsin-stable triple helices and were secreted efficiently from the cell. The thermal stability of wild-type molecules and molecules containing the reporter [Ile822]alpha 1(I) chains was identical. The biosynthetic characteristics of wild-type and reporter chains were directly compared in stably transfected 3T6 cells. These cells did not make a distinction between reporter and endogenous alpha 1(I) chains, which were secreted from the cells at the same rate and were processed and deposited into the 3T6 cell in vitro accumulated extracellular matrix with equal efficiency. These data demonstrate that the helical sequence alteration in the reporter protein is functionally neutral and that the reporter construct, pWTCI-Ile822, is a suitable vector for the analysis of the biochemical effects of site-directed mutations in the putative COL1A1 functional domains.

Amino Acid Sequence↗
PubMed1997

Modified substrate specificity of L-hydroxyisocaproate dehydrogenase derived from structure-based protein engineering.

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad substrate specificity and utilizes a wide range of 2-oxo acids branched at the C4 atom. Modifications have been made to the sequence of the NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and alter the region of substrate specificity towards various 2-oxocarbonic acids. All variations were based on a 3D-structure model of the enzyme using the X-ray coordinates of the functionally related L-lactate dehydrogenase (L-LDH) from dogfish as a template. This protein displays only 23% sequence identity to L-HicDH. The active site of L-HicDH was modelled by homology to the L-LDH based on the conservation of catalytically essential residues. Substitutions of the active site residues Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify their unique participation in substrate recognition and orientation. The kinetic properties of the L239A, L239M, L236V and T245A enzyme variants confirmed the structural model of the active site of L-HicDH. The substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate, phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the active site of the subsequently refined model. In order to design dehydrogenases with an improved substrate specificity towards keto acids branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236 and Thr245 were introduced and resulted in mutant enzymes with completely different substrate specificities. The substitution T245A resulted in a relative shift of substrate specificity for keto-tert-leucine of more than 17000 compared with the 2-oxocaproate (kcat/KM). For the substrates branched at C4 a relative shift of up to 500 was obtained for several enzyme variants. A total of nine mutations were introduced and the kinetic data for the set of six substrates were determined for each of the resulting mutant enzymes. These were compared with those of the wild-type enzyme and rationalized by the active site model of L-HicDH. An analysis of the enzyme variants provided new insight into the residues involved in substrate binding and residues of importance for the differences between LDHs and HicDH. After the protein design project was complete the X-ray structure of the enzyme was solved in our group. A comparison between the model and the experimental 3D structure proved the quality of the model. All the variants were designed, expressed and tested before the 3D structure became available.

Alcohol Oxidoreductases↗