Optical studies of single-tryptophan B. licheniformis ¦Â-lactamase variants

VALERIA A. RISSO; MAR¨ªA E. PRIMO; JUAN E. BRUNET; CARLOS P. SOTOMAYOR; MARIO R. ERM¨¢CORA

BIOPHYSICAL CHEMISTRY

ELSEVIER SCIENCE BV

Año: 2010 vol. 151 p. 111 - 118

0301-4622

¦Â-lactamases (penicillinases) are important complicating factors in bacterial infections and excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A ¦Â-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp¡úPhe mutantswere prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolved-lactamases (penicillinases) are important complicating factors in bacterial infections and excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A ¦Â-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp¡úPhe mutantswere prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolvedBacillus licheniformis exo-small penicillinase (ESP) is a Class A ¦Â-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp¡úPhe mutantswere prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolved¦Â-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp¡úPhe mutantswere prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolvedfluorescence, four Trp¡úPhe mutantswere prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolvedfied and ascribed to specific aromatic residues. Time-resolved fluorescence showed that most of the fluorescence decay of ESP tryptophans is due to a discrete exponential componentwith a lifetime of 5¨C6 ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251,whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A ¦Â-lactamases.uorescence showed that most of the fluorescence decay of ESP tryptophans is due to a discrete exponential componentwith a lifetime of 5¨C6 ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251,whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A ¦Â-lactamases.¨C6 ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251,whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A ¦Â-lactamases.fluorescence of Trp 229 and Trp 251,whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A ¦Â-lactamases.fluctuations of ESP and other Class A ¦Â-lactamases.