PDBsum entry 4bcs

Biotin-binding protein

PDB id: 4bcs

Contents

Protein chains

123 a.a.

Ligands

ACT

PEG

BTN ×2

PO4 ×2

Waters ×112

PDB id:

4bcs

Name:

Biotin-binding protein

Title:

Crystal structure of an avidin mutant

Structure:

Chimeric avidin. Chain: a, b. Engineered: yes

Source:

Gallus gallus. Chicken. Organism_taxid: 9031. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.80Å R-factor: 0.191 R-free: 0.226

Authors:

T.T.Airenne,B.Niederhauser,V.P.Hytonen,M.S.Kulomaa,M.S.Johnson

Key ref:

B.Taskinen et al. (2014). A novel chimeric avidin with increased thermal stability using DNA shuffling. Plos One, 9, e92058. PubMed id: 24632863 DOI: 10.1371/journal.pone.0092058

Date:

03-Oct-12 Release date: 16-Oct-13

Protein chains

P56734  (AVR4_CHICK) -  Avidin-related protein 4/5 from Gallus gallus

Seq: 150 a.a.

Struc: 123 a.a.*

* PDB and UniProt seqs differ at 15 residue positions (black crosses)

DOI no: 10.1371/journal.pone.0092058

Plos One 9:e92058 (2014)

PubMed id: 24632863

A novel chimeric avidin with increased thermal stability using DNA shuffling.

B.Taskinen, T.T.Airenne, J.Jänis, R.Rahikainen, M.S.Johnson, M.S.Kulomaa, V.P.Hytönen.

ABSTRACT

Avidins are a family of proteins widely employed in biotechnology. We have previously shown that functional chimeric mutant proteins can be created from avidin and avidin-related protein 2 using a methodology combining random mutagenesis by recombination and selection by a tailored biopanning protocol (phage display). Here, we report the crystal structure of one of the previously selected and characterized chimeric avidin forms, A/A2-1. The structure was solved at 1.8 Å resolution and revealed that the protein fold was not affected by the shuffled sequences. The structure also supports the previously observed physicochemical properties of the mutant. Furthermore, we improved the selection and screening methodology to select for chimeric avidins with slower dissociation rate from biotin than were selected earlier. This resulted in the chimeric mutant A/A2-B, which showed increased thermal stability as compared to A/A2-1 and the parental proteins. The increased stability was especially evident at conditions of extreme pH as characterized using differential scanning calorimetry. In addition, amino acid sequence and structural comparison of the chimeric mutants and the parental proteins led to the rational design of A/A2-B I109K. This mutation further decreased the dissociation rate from biotin and yielded an increase in the thermal stability.