Biomedical subjects

Malin Allert

Publications and source records attributed to Malin Allert.

4 recordsLinked to original sources

PubMed2006

Local encoding of computationally designed enzyme activity.

One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously, we showed that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here, we report that these mutations can be "transplanted" into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 10(5)-10(6)-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is encoded locally within the layer of residues directly in contact with the substrate and that, in this case, at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants.

Amino Acid Sequence↗

PubMed2004

Computational design of receptors for an organophosphate surrogate of the nerve agent soman.

We report the computational design of soluble protein receptors for pinacolyl methyl phosphonic acid (PMPA), the predominant hydrolytic product of the nerve agent soman. Using recently developed computational protein design techniques, the ligand-binding pockets of two periplasmic binding proteins, glucose-binding protein and ribose-binding protein, were converted to bind PMPA instead of their cognate sugars. The designs introduce 9-12 mutations in the parent proteins. Twelve of 20 designs tested exhibited PMPA-dependent changes in emission intensity of a fluorescent reporter with affinities between 45 nM and 10 microM. The contributions to ligand binding by individual residues were determined in two designs by alanine-scanning mutagenesis, and are consistent with the molecular models. These results demonstrate that designed receptors with radically altered binding specificities and affinities that rival or exceed those of the parent proteins can be successfully predicted. The designs vary in parent scaffold, sequence diversity, and orientation of docked ligand, suggesting that the number of possible solutions to the design problem is large and degenerate. This observation has implications for the genesis of biological function by random processes. The designed receptors reported here may have utility in the development of fluorescent biosensors for monitoring nerve agents.

Binding Sites↗

PubMed2003

Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif-implications for catalyst design.

In our search for a catalyst for the transamination reaction of aspartic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reaction that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18500 deg x cm(2) dmol(-1). Upon addition of 6 mm l-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19900 deg x cm(2) dmol(-1). The corresponding mean residue ellipticities of T-16 were -21200 deg x cm(2) dmol(-1) and -24000 deg x cm(2) dmol(-1). These results show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The (1)H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the (31)P NMR resonance linewidth. The catalytic efficiencies of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show for the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.

Amino Acid Sequence↗

PubMed2002

Setting the stage for new catalytic functions in designed proteins--exploring the imine pathway in the efficient decarboxylation of oxaloacetate by an Arg-Lys site in a four-helix bundle protein scaffold.

Fourteen 42-residue polypeptides have been designed to identify reactive sites for the catalysis of the decarboxylation of oxaloacetate, a chemical transformation that proceeds through the formation of an imine intermediate. The sequences fold into helix-loop-helix motifs and dimerize to four-helix bundles. The catalytically active lysine residues were incorporated in several surface exposed positions, but also in positions characterised by hydrophobic properties to reduce their pKa values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilise and bind the imine intermediate in the decarboxylation reaction. A two-residue Arg-Lys site formed the main component of the reactive site of the helix-loop-helix dimer Decarb-K34_R33, which obeyed saturation kinetics in catalysing the reaction with a kcat/KM of 0.59 M-1S-1. The rate constant measured was nearly three orders of magnitude larger than the second-order rate constant of the butylamine-catalysed reaction (0.0011 M-1S-1), and four orders of magnitude larger than the pseudo first-order rate constant of the uncatalyzed reaction (1.3 x 10(-5) s(-1)). The sequence of Decarb-K34_R33 contained only a single lysine residue. It was flanked by an arginine in the preceding position in the sequence. A flanking Arg residue provided more efficient catalysis than a flanking Lys or Gln residue. Arginines in flanking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in catalysis as the Arg of the Arg-Lys pair. The effect of pKa on the catalytic efficiency of the Lys residue in the decarboxylation reaction is well known. The identification of the role of the flanking Arg residue in catalysing decarboxylation, its optimal position, and the importance of conformational stability reported here sets the stage for developing a number of catalytic systems that depend on the formation of imine intermediates, but that lead to different reaction products.

Amino Acid Sequence↗