Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin.
Artikel i vetenskaplig tidskrift, 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.

Azurin

chemistry

genetics

Zinc

genetics

Cysteine

Calorimetry

chemical synthesis

Electrostatics

genetics

Thermodynamics

Pseudomonas aeruginosa

Lysine

X-Ray

Spectrophotometry

chemistry

genetics

Mass

genetics

chemistry

Differential Scanning

Arginine

Crystallography

chemistry

genetics

Site-Directed

Mutagenesis

Electrospray Ionization

Disulfides

Aspartic Acid

Mass Spectrometry

Protein Denaturation

genetics

Copper

Spectrometry

Författare

ANNA KATARINA TIGERSTRÖM

Göteborgs universitet

Frederick Schwarz

National Institute of Standards and Technology

B Göran Karlsson

Chalmers, Institutionen för kemi och biovetenskap, Molekylär bioteknik

M. Ökvist

Göteborgs universitet

Carmen Alvarez-Rúa

Universidad de Oviedo

Dennis Maeder

UMBI

Frank T Robb

UMBI

Lennart Sjölin

Göteborgs universitet

Biochemistry

0006-2960 (ISSN) 1520-4995 (eISSN)

Vol. 43 39 12563-74

Ämneskategorier

Kemi

DOI

10.1021/bi048926x

PubMed

15449946

Mer information

Skapat

2017-10-08