X‐ray evidence of a native state with increased compactness populated by tryptophan‐less B. licheniformis β‐lactamase

Abstract

b-lactamases confer antibiotic resistance, one of the most serious world-wide health problems, and are an excellent theoretical and experimental model in the study of protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class-A b-lactamase with three tryptophan residues located in the protein core. Here, we report the 1.7-Å resolution X-ray structure, catalytic parameters, and thermodynamic stability of ESP DW , an engineered mutant of ESP in which phenylalanine replaces the wild-type tryptophan residues. The structure revealed no qualitative conformational changes compared with thirteen previously reported structures of B. licheniformis b-lactamases (RMSD 5 0.4-1.2 Å ). However, a closer scrutiny showed that the mutations result in an overall more compact structure, with most atoms shifted toward the geometric center of the molecule. Thus, ESP DW has a significantly smaller radius of gyration (R g ) than the other B. licheniformis b-lactamases characterized so far. Indeed, ESP DW has the smallest R g among 126 Class-A b-lactamases in the Protein Data Bank (PDB). Other measures of compactness, like the number of atoms in fixed volumes and the number and average of noncovalent distances, confirmed the effect. ESP DW proves that the compactness of the native state can be enhanced by protein engineering and establishes a new lower limit to the compactness of the Class-A blactamase fold. As the condensation achieved by the native state is a paramount notion in protein folding, this result may contribute to a better understanding of how the sequence determines the conformational variability and thermodynamic stability of a given fold.

Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins

“…Protein purity was checked by SDS‐PAGE. Lactamase activity in cellular extracts after induction was assessed by following spectrophotometrically (486 nm) the degradation of nitrocefin . Enzyme kinetic parameters ( K m , k cat and k cat / K m ) for the lactamase‐catalyzed hydrolysis of benzylpenicillin (BZ), cefotaxime (CTX), and ceftazidime (CAZ) at 25°C in 50 m M sodium phosphate buffer pH 7.0 were determined from the fitting of the Michaelis‐Menten equation to profiles of initial rate versus substrate concentration (see Fig.…”

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

“…In order to fully exploit the advantages of site-specific Trp fluorescence, the intrinsic Pgp fluorescence must be reduced or eliminated before strategically placed Trps can be used to localize the discrete drug binding sites and to measure binding affinities for drugs and ATP. A number of proteins [56][57][58][59][60][61][62][63][64][65][66] , including some membrane proteins [67][68][69][70][71][72] , have been rendered 36 with the N-and C-terminal halves of the protein colored in blue and green, respectively. Pgp has eight Trps in the transmembrane domains (TMDs), and three Trps in the cytoplasmic domains shown as red spheres and labeled with residue numbers.…”