Interactions Between Small Heat Shock Proteins and Substrate Proteins Mass Spectrometric Detection of Crosslinked Peptides to Map Protein-Protein Interactions

Research output: ThesisDoctoral Thesis (compilation)

Abstract

The small heat shock proteins (sHsps) are a widespread class of molecular chaperones existing in all organisms. During heat stress the

sHsps protect other proteins from unfolding and aggregation. In plants the sHsps play an especially prominent part of the stress

response. In this thesis chemical crosslinking and mass spectrometric mapping of crosslinked peptides has been used to investigate the

interaction between sHsps and substrate proteins. The focus has been on Hsp21, a chloroplast-localized sHsp which increases the stress

resistance of Arabidopsis thaliana plants, and a thermosensitive model substrate protein, citrate synthase (CS), using the chemical

crosslinker DTSSP (3,3?-dithiobis[sulfosuccinimidylpropionate]), for covalent crosslinking between lysine residues or other primary

amines.

In peptide array screening, binding of sHsps occurred to a peptide located in a stem-like structure protruding from the CS dimer, where

the C-terminus from one CS monomer interacts with the N-terminal part of the other monomer. Mass spectrometric mapping of

crosslinked Hsp21-CS peptides further confirmed interactions between Hsp21 and lysine residues (K16, K22, K432, K437) located in

this stem-like structure of CS. We propose that sHsps bind to this region, which is absent in thermostable CS forms, to stabilize the Nand

C-terminae and thereby prevent CS dimer dissociation and aggregation.

The crosslinked Hsp21-CS peptides indicated that the substrate-binding region in the N-terminal arm in Hsp21 interacted with the

thermosensitive part of CS. Similar mass spectrometric peptide mapping was also used to show how this N-terminal arm region of a

mammalian sHsp, Hsp20, is involved in transglutaminase-catalyzed crosslinking of sHsps to various substrate proteins in cellular

aggregates. This was studied using a peptide probe which could be crosslinked by transglutaminase to the conserved Q31 in the Nterminal

arm of mammalian sHsps.

Whereas the N-terminus of Hsp21 was detected in crosslinked Hsp21-Hsp21 and Hsp21-CS peptides under conditions where the

Hsp21 dodecamer was intact, the C-terminal tail and a C-terminal binding groove was detected in crosslinked Hsp21-CS peptides only

under conditions where the Hsp21 dodecamer dissociated. The crosslinked Hsp21-CS peptides mapped onto several sites on the CS

surface, indicating that several weak and short-lived interactions between Hsp21 and CS occur already at normal temperatures. Single

particle reconstruction EM of crosslinked Hsp21 in the presence of CS indicated that the crosslinker could capture the Hsp21

dodecamers in an ?activated? conformation induced by such weak and short-lived interactions with CS.

Details

Authors
  • Emma Åhrman
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Chemical Sciences

Keywords

  • Protein-protein interactions, Small heat shock proteins, Mass spectrometric peptide mapping, Proteins, enzymology, Proteiner, enzymologi
Original languageEnglish
QualificationDoctor
Awarding Institution
Supervisors/Assistant supervisor
Award date2007 Jun 9
Publisher
  • Department of Biochemistry, Lund University
Print ISBNs978-91-7422-162-6
Publication statusPublished - 2007
Publication categoryResearch

Bibliographic note

Defence details Date: 2007-06-09 Time: 09:15 Place: Lecture hall B Centre for Chemistry and Chemical Engineering K Getingevägen 60 22241 Lund External reviewer(s) Name: Kampinga,, Harm Title: Professor Affiliation: Department of Cell Biology, Section of Radiation and Stress Cell Biology, University of Groningen, T --- The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Center for Chemistry and Chemical Engineering (011001000), Biochemistry and Structural Biology (S) (000006142)