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The Protein Journal

, Volume 31, Issue 1, pp 93–107 | Cite as

Expression, Purification, Structural and Functional Analysis of SycB: A Type Three Secretion Chaperone From Yersinia enterocolitica

  • Abhishek Basu
  • Rakesh Chatterjee
  • Saumen Datta
Article

Abstract

In Yersinia enterocolitica biovar 1B, a genome encoded TTSS designated as Ysa-Ysp system is used for virulence. SycB is an annotated chaperone to this system. SycB is soluble in presence of translocator YspC. SycB and its truncated form (∆SycB(1–114)) exist as dimers. YspC forms a 1:1 complex with SycB. Homology model of SycB shows a flexible N-terminal may be required for solubility and dimerization; and concave core formed by antiparallel helices of TPRs. Far UV CD spectra confirm that SycB is predominantly alpha helical. Near UV CD spectra show that SycB has tertiary structure at pH 7.2 (native folded protein), which disappears at pH 5 (molten globule) and SycB releases YspC at pH 5. SycB has a cooperative melting behavior. At pH 7.2, SycB shows solvent accessible hydrophobic patches. Concave core in the model exhibits ANS binding within FRET distance of tyrosines in the TPR, allowing a range of interaction of SycB with its ligand.

Keywords

Class II chaperone Homology model TPR helices pH and protein folding Molten Globule Intrinsic and extrinsic fluorophore ANS quenching and FRET 

Abbreviations

TTSS

Type three secretion system

Ysa-Ysp

Yersinia secretion apparatus-Yersinia secretion protein

TPR

Tetratricopeptide repeat

CD

Circular dichroism

ANS

8-anilinonapthalene-1-sulfonate

FRET

Forsters resonance energy transfer

Ysc-Yop

Yersinia secretion component-Yersinia outer protein

DLS

Dynamic light scattering

BLAST

Basic Local Search Alignment Tool

NCBI

National Center for Biotechnology Information

MSA

Multiple sequence alignment

PDB

Protein Data Bank

ATCC

American Type Culture Collection

MCS

Multiple cloning site

LB

Luria Bertani

IPTG

Isopropyl β-D-1-thiogalactopyranoside

PMSF

Phenylmethanesulfonyl fluoride

Ni–NTA

Nickel–nitrilotriacetic acid

CHCA

α-cyano hydroxyl cinnamic acid

MALDI

Matrix assisted laser desorption ionization

EDTA

Ethylenediaminetetraacetic acid

EGS

Ethylene Glycol bis[succinimidyl succinate]

TOF

Time of flight

Notes

Acknowledgments

The research was funded by grants from the Department of Science and Technology, Government of India. Indian Institute of Chemical Biology (IICB), a unit of Council of Scientific and Industrial Research provided the facilities and fellowships for this research.

Supplementary material

10930_2011_9377_MOESM1_ESM.doc (166 kb)
Supplementary material 1 (DOC 166 kb)

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Abhishek Basu
    • 1
  • Rakesh Chatterjee
    • 1
  • Saumen Datta
    • 1
  1. 1.Structural Biology and Bioinformatics DivisionIndian Institute of Chemical BiologyKolkataIndia

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