The conserved methionines in the chloroplast small heat shock protein - Role in chaperone-substrate interactions and effects of methionine sulfoxidation on chaperone activity
(2001)- Abstract
- The chloroplast-localized small heat shock protein (sHsp) Hsp21 belongs to the family of alpha-crystallin like sHsps which form large oligomeric structures and protect partly unfolded aggregation-prone proteins against aggregation. The main focus for this thesis has been on the highly conserved methionines in Hsp21. These methionine residues are situated on one side of an amphipathic alpha-helix. Methionines are easily oxidized which leads to dramatical changes in the biophysical properties of the methionine side chain. The dependence of the conserved methionines for Hsp21 chaperone-like activity has been studied and the impact of methionine sulfoxidation on Hsp21 structure and function.
Direct analysis of the... (More) - The chloroplast-localized small heat shock protein (sHsp) Hsp21 belongs to the family of alpha-crystallin like sHsps which form large oligomeric structures and protect partly unfolded aggregation-prone proteins against aggregation. The main focus for this thesis has been on the highly conserved methionines in Hsp21. These methionine residues are situated on one side of an amphipathic alpha-helix. Methionines are easily oxidized which leads to dramatical changes in the biophysical properties of the methionine side chain. The dependence of the conserved methionines for Hsp21 chaperone-like activity has been studied and the impact of methionine sulfoxidation on Hsp21 structure and function.
Direct analysis of the methionione sulfoxide content of Hsp21 was performed by MALDI/TOF mass spectrometry. The methionine sulfoxidation was found to occur concomitantly with a change in the oligomeric conformation and loss of chaperone-like activity, both probably due to loss of alpha-helical structure in the methionine-rich region. By construction of Hsp21 mutants it was concluded that leucines can replace methionines in terms of chaperone-like activity. Furthermore, it was discovered that methionine sulfoxides in Hsp21 were reducible by a chloroplast-localized form of peptide methionine sulfoxide (pPMSR) and Hsp21, with its many oxidizable methionines is thus a likely endogenous substrate for pPMSR. Hence, pPMSR provides a means for keeping the methionines in Hsp21 in their reduced state and therby protection of Hsp21 chaperone-like activity in vivo. By using peptide library arrays map the sites of interaction between the methionine-rich region in Hsp21 and at least two different regions in the model substrate protein citrate synthase (CS), probably reflecting a differential chaperone-like activity of Hsp21. One of these regions belongs to a surface exposed alpha-helix in porcine CS. This alpha-helix is absent in CS from the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus.
The data presented in this thesis point to a role of the methionines in substrate binding, that pPMSR co-evolved with Hsp21, that methionines may be preferred over leucines due to structural flexibility and efficient subunit exchange and that methionine sulfoxidation may play a role in regulation. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
I gröna växters kloroplaster finns ett s.k. sHsp-protein (small Heat shock protein) som betecknas Hsp21. Det tillhör en grupp av hjälpar-protein som kallas molekylära chaperoner. Chaperoner hjälper andra proteiner så att de inte klibbar ihop, aggregerar och blir inaktiva. Två vattenskyende ytor som sammanförs aggregerar. Genom att särskilt aggregerings-benägna (vattenskyende) ytor i proteinerna fästs upp mot chaperonets yta skyddas de från att aggregera. Denna avhandling fokuserar på en särskild sorts aminosyra som förekommer rikligt i Hsp21, aminosyran metionin. Under evolutionen då de gröna växterna utvecklats för ett liv på land har metioniner bevarats i proteinet. Metioninerna bildar... (More) - Popular Abstract in Swedish
I gröna växters kloroplaster finns ett s.k. sHsp-protein (small Heat shock protein) som betecknas Hsp21. Det tillhör en grupp av hjälpar-protein som kallas molekylära chaperoner. Chaperoner hjälper andra proteiner så att de inte klibbar ihop, aggregerar och blir inaktiva. Två vattenskyende ytor som sammanförs aggregerar. Genom att särskilt aggregerings-benägna (vattenskyende) ytor i proteinerna fästs upp mot chaperonets yta skyddas de från att aggregera. Denna avhandling fokuserar på en särskild sorts aminosyra som förekommer rikligt i Hsp21, aminosyran metionin. Under evolutionen då de gröna växterna utvecklats för ett liv på land har metioniner bevarats i proteinet. Metioninerna bildar tillsammans en särskild yta i Hsp21-proteinet, ytan på en helix (=spiral). Spiralen är vattenskyende på ena sidan, och vattenälskande på den andra. Om metionerna utsätts för oxidation förändras deras biofysikaliska egenskaper drastiskt.
I avhandlingen har jag studerat vilken betydelse metioninerna har för att Hsp21 ska kunna verka som ett molekylärt chaperon och hjälpa och förhindra att andra proteiner aggregerar, och vad som händer om metioninerna i Hsp21 råkar ut för oxidation av tex det syre som naturligt produceras i kloroplasten. För att mäta hur mycket oxiderat metionin som finns i Hsp21 under olika betingelser användes mass spektrometri, en avancerad metod som kan uppskatta vikten på olika molekyler väldigt exakt. Jag fann att om metioninerna oxiderades så förändrades formen på Hsp21-proteinet, samtidigt som spiralen upplöstes och förmågan att verka som ett molekulärt chaperon försvann. Om aminosyran metionin byttes ut mot den oxidations-okänsliga aminosyran leucin genom s.k. riktad mutagenes förblev Hsp21 fortfarande verksamt som chaperon, men känsligheten för oxidation var borta. I avhandlingen visas också hur ett annat protein i kloroplasten, PMSR, i sin tur kan hjälpa hjälpar-proteinet Hsp21 genom att reparera dess metioniner om de blivit oxiderade. Tack vare PMSR kan alltså Hsp21 ständigt hållas i bra skick och vara verksamt som chaperon. Slutligen har jag i avhandlingen påbörjat en undersökning av hur Hsp21 hjälper andra protein: med ett s.k. peptid bibliotek kunde vi se vilka regioner i enzymet citrat-syntas som skyddades mot aggregering genom att fästas upp på Hsp21, troligen mot den metionin-innehållande spiralen. Citrat-syntas är ett enzym som finns i praktiskt taget alla levande organismer. Just de ytor som fästes upp mot Hsp21 visade sej vara en särskilt temperaturkänslig region i citrat syntas. Bakterier som anpassats till att leva vid mycket höga temperaturer i tex varma källor saknar i sitt citrat syntas helt och hållet denna känsliga region.
Sammanfattningvis pekar de data som presenteras i denna avhandling på att metioninerna behövs för att bilda den spiral mot vilken proteiner fästs upp för att inte aggregera. Eftersom PMSR-proteinet kontinuerligt kan reparera oxidations-skadade metioniner tycks fördelarna med aminosyran metionin framför leucin överväga, kanske är det tom så att viss oxidation behövs för reglering av Hsp21. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/41903
- author
- Gustavsson, Niklas LU
- supervisor
- opponent
-
- Bukau, Bernd
- organization
- publishing date
- 2001
- type
- Thesis
- publication status
- published
- subject
- keywords
- Metabolism, Biochemistry, oxidative stress, thermostability, temperature stress, stress tolerance, sHsp, protein aggregation, molecular chaperone, Heat stress, methionine sulfoxidation, Biokemi, metabolism, Plant biochemistry, Växtbiokemi
- pages
- 164 pages
- publisher
- Department of Biochemistry, Lund University
- defense location
- Lecturehall C, Center for Chemistry and Chemical Engeneering, Lund, Sweden
- defense date
- 2001-10-19 10:15:00
- external identifiers
-
- other:ISRN: LUNDKL/(NKBK-1071)/1-164/2001
- ISBN
- 91-628-4982-4
- language
- English
- LU publication?
- yes
- id
- 4d8dd24a-a410-478e-8d55-5da5cc3b3bf1 (old id 41903)
- date added to LUP
- 2016-04-04 10:40:12
- date last changed
- 2018-12-10 13:56:33
@phdthesis{4d8dd24a-a410-478e-8d55-5da5cc3b3bf1,
abstract = {{The chloroplast-localized small heat shock protein (sHsp) Hsp21 belongs to the family of alpha-crystallin like sHsps which form large oligomeric structures and protect partly unfolded aggregation-prone proteins against aggregation. The main focus for this thesis has been on the highly conserved methionines in Hsp21. These methionine residues are situated on one side of an amphipathic alpha-helix. Methionines are easily oxidized which leads to dramatical changes in the biophysical properties of the methionine side chain. The dependence of the conserved methionines for Hsp21 chaperone-like activity has been studied and the impact of methionine sulfoxidation on Hsp21 structure and function.<br/><br>
<br/><br>
Direct analysis of the methionione sulfoxide content of Hsp21 was performed by MALDI/TOF mass spectrometry. The methionine sulfoxidation was found to occur concomitantly with a change in the oligomeric conformation and loss of chaperone-like activity, both probably due to loss of alpha-helical structure in the methionine-rich region. By construction of Hsp21 mutants it was concluded that leucines can replace methionines in terms of chaperone-like activity. Furthermore, it was discovered that methionine sulfoxides in Hsp21 were reducible by a chloroplast-localized form of peptide methionine sulfoxide (pPMSR) and Hsp21, with its many oxidizable methionines is thus a likely endogenous substrate for pPMSR. Hence, pPMSR provides a means for keeping the methionines in Hsp21 in their reduced state and therby protection of Hsp21 chaperone-like activity in vivo. By using peptide library arrays map the sites of interaction between the methionine-rich region in Hsp21 and at least two different regions in the model substrate protein citrate synthase (CS), probably reflecting a differential chaperone-like activity of Hsp21. One of these regions belongs to a surface exposed alpha-helix in porcine CS. This alpha-helix is absent in CS from the thermophilic archaea Thermoplasma acidophilum and Pyrococcus furiosus.<br/><br>
<br/><br>
The data presented in this thesis point to a role of the methionines in substrate binding, that pPMSR co-evolved with Hsp21, that methionines may be preferred over leucines due to structural flexibility and efficient subunit exchange and that methionine sulfoxidation may play a role in regulation.}},
author = {{Gustavsson, Niklas}},
isbn = {{91-628-4982-4}},
keywords = {{Metabolism; Biochemistry; oxidative stress; thermostability; temperature stress; stress tolerance; sHsp; protein aggregation; molecular chaperone; Heat stress; methionine sulfoxidation; Biokemi; metabolism; Plant biochemistry; Växtbiokemi}},
language = {{eng}},
publisher = {{Department of Biochemistry, Lund University}},
school = {{Lund University}},
title = {{The conserved methionines in the chloroplast small heat shock protein - Role in chaperone-substrate interactions and effects of methionine sulfoxidation on chaperone activity}},
year = {{2001}},
}