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.

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

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

Senast uppdaterat

2019-05-23