Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin.
Journal article, 2004
Identification and evaluation of factors important for thermostability in proteins is a growing research field with many industrial applications. This study investigates the effects of introducing a novel disulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurin from Pseudomonas aeruginosa. Four mutants were selected on the basis of rational design and novel temperature-dependent atomic displacement factors from crystal data collected at elevated temperatures. The atomic displacement parameters describe the molecular movement at higher temperatures. The thermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry. Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting a flexible loop with small alpha-helix (D62C/K74C copper-containing mutant), increased the T(m) by 3.7 degrees C compared with the holo protein. Furthermore, three mutants were designed to introduce electrostatic interactions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between two separate beta-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore, D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability. The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to 1.5 and 1.8 A resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has also been completed to 1.8 A resolution. These structures support the selected design and provide valuable information for evaluating effects of the modifications on the thermostability of holo azurin.
Spectrophotometry
Cysteine
Site-Directed
genetics
Aspartic Acid
genetics
Calorimetry
chemical synthesis
Azurin
chemistry
Thermodynamics
Zinc
chemistry
Lysine
Mass Spectrometry
Arginine
Crystallography
genetics
Spectrometry
Copper
Protein Denaturation
Electrostatics
chemistry
X-Ray
chemistry
Disulfides
Differential Scanning
genetics
Mass
genetics
genetics
Pseudomonas aeruginosa
Mutagenesis
Electrospray Ionization
genetics
Author
ANNA KATARINA TIGERSTRÖM
University of Gothenburg
Frederick Schwarz
National Institute of Standards and Technology (NIST)
B Göran Karlsson
Chalmers, Department of Chemistry and Bioscience, Molecular Biotechnology
M. Ökvist
University of Gothenburg
Carmen Alvarez-Rúa
University of Oviedo
Dennis Maeder
UMBI
Frank T Robb
UMBI
Lennart Sjölin
University of Gothenburg
Biochemistry
0006-2960 (ISSN) 1520-4995 (eISSN)
Vol. 43 39 12563-74Subject Categories
Chemical Sciences
DOI
10.1021/bi048926x
PubMed
15449946