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NMR studies of the pH effect on the conformational exchange of enzyme FKBP12

Wernersson, Sven LU (2016) KFK920 20161
Biophysical Chemistry
Abstract
This master thesis project reports the dependence on pH of the conformational exchange in the enzyme FKBP12. Previous NMR studies have identified two conformational exchange processes in FKBP12, where the faster process is believed to be an exchange to an intermediate state between the more slowly exchanging conformations.
In this project I carried out different NMR experiments across the pH-intervals 5-7 and 5-8. I used 15N and 13C HSQC and CPMG experiments to monitor chemical shift changes and characterize conformational exchange as a function of pH.
For the faster process it was found that as the pH decreases, the major exchange conformation appears to be more stabilized relative to both the minor state of the fast exchange process... (More)
This master thesis project reports the dependence on pH of the conformational exchange in the enzyme FKBP12. Previous NMR studies have identified two conformational exchange processes in FKBP12, where the faster process is believed to be an exchange to an intermediate state between the more slowly exchanging conformations.
In this project I carried out different NMR experiments across the pH-intervals 5-7 and 5-8. I used 15N and 13C HSQC and CPMG experiments to monitor chemical shift changes and characterize conformational exchange as a function of pH.
For the faster process it was found that as the pH decreases, the major exchange conformation appears to be more stabilized relative to both the minor state of the fast exchange process and to the energy barrier between them. For the slower process there were no notable change in the free energy of the barrier between the states of the slow exchange process, while the free energy difference to the minor state of the slow exchange process appeared to decrease with a decrease in pH.
In addition to the findings directly connected to the main processes, it was found that the inherent flexibility of Gly89 appears to increase with a decrease in pH. Also, below pH 6 the CPMG dispersions from multiple residues show increased conformational exchange, which could be due to the appearance of new exchange processes. (Less)
Popular Abstract (Swedish)
I detta examensarbete studeras inverkan av pH på konformationsprocesserna i enzym FKBP12 med hjälp av kärnmagnetisk resonansspektroskopi (NMR). Tidigare studier har identifierat två konformationsprocesser i FKBP12, där den mindre befolkade konformationen i den snabbare processen misstänks vara en intermediär konformation mellan konformationerna som deltar i den långsammare processen.
I projektet genomförde jag HSQC och CPMG NMR-experiment i pH intervallen 5-7 och 5-8, för både 15N och 13C. Detta för att följa förändringar i det kemiska skiftet, samt karakterisera konformationsprocesserna vid olika pH.
Resultatet för den snabbare processen var att den mest befolkade konformationen tycks stabiliseras relativt den andra konformationen vid... (More)
I detta examensarbete studeras inverkan av pH på konformationsprocesserna i enzym FKBP12 med hjälp av kärnmagnetisk resonansspektroskopi (NMR). Tidigare studier har identifierat två konformationsprocesser i FKBP12, där den mindre befolkade konformationen i den snabbare processen misstänks vara en intermediär konformation mellan konformationerna som deltar i den långsammare processen.
I projektet genomförde jag HSQC och CPMG NMR-experiment i pH intervallen 5-7 och 5-8, för både 15N och 13C. Detta för att följa förändringar i det kemiska skiftet, samt karakterisera konformationsprocesserna vid olika pH.
Resultatet för den snabbare processen var att den mest befolkade konformationen tycks stabiliseras relativt den andra konformationen vid låga pH. För den långsamma processen var resultatet det motsatta, dvs att den mindre befolkade konformationen stabiliserades vid lågt pH.
Förutom resultaten för de tidigare karakteriserade processerna framkom det att den inneboende flexibiliteten hos glycin 89 tycks öka vid minskat pH. Dessutom så visade CPMG dispersionerna för flera toppar under pH 6 tecken på en ny konformationsprocess. (Less)
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author
Wernersson, Sven LU
supervisor
organization
course
KFK920 20161
year
type
H2 - Master's Degree (Two Years)
subject
keywords
FKBP12, ISOMERASE, NUCLEAR MAGNETIC RESONANCE, NMR, CPMG, BIOPHYSICAL CHEMISTRY, CONFORMATIONAL EXCHANGE, BIOFYSIKALISK KEMI, KÄRNMAGNETISK RESONANS, ISOMERAS
language
English
id
8883866
date added to LUP
2016-06-28 11:05:15
date last changed
2017-03-03 15:04:33
@misc{8883866,
  abstract     = {This master thesis project reports the dependence on pH of the conformational exchange in the enzyme FKBP12. Previous NMR studies have identified two conformational exchange processes in FKBP12, where the faster process is believed to be an exchange to an intermediate state between the more slowly exchanging conformations. 
In this project I carried out different NMR experiments across the pH-intervals 5-7 and 5-8. I used 15N and 13C HSQC and CPMG experiments to monitor chemical shift changes and characterize conformational exchange as a function of pH. 
For the faster process it was found that as the pH decreases, the major exchange conformation appears to be more stabilized relative to both the minor state of the fast exchange process and to the energy barrier between them. For the slower process there were no notable change in the free energy of the barrier between the states of the slow exchange process, while the free energy difference to the minor state of the slow exchange process appeared to decrease with a decrease in pH. 
In addition to the findings directly connected to the main processes, it was found that the inherent flexibility of Gly89 appears to increase with a decrease in pH. Also, below pH 6 the CPMG dispersions from multiple residues show increased conformational exchange, which could be due to the appearance of new exchange processes.},
  author       = {Wernersson, Sven},
  keyword      = {FKBP12,ISOMERASE,NUCLEAR MAGNETIC RESONANCE,NMR,CPMG,BIOPHYSICAL CHEMISTRY,CONFORMATIONAL EXCHANGE,BIOFYSIKALISK KEMI,KÄRNMAGNETISK RESONANS,ISOMERAS},
  language     = {eng},
  note         = {Student Paper},
  title        = {NMR studies of the pH effect on the conformational exchange of enzyme FKBP12},
  year         = {2016},
}