An investigation of the mechanical properties of the molten globule state of apomyoglobin

Single molecule force spectroscopy has provided important insights into the properties and mechanisms of protein folding. However, there are still many unanswered questions about how force affects the folding and unfolding of proteins and, in particular, the relationship between force and the rate-l...

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Main Author: Elms, Phillip James
Other Authors: Marqusee, Susan
Format: Other/Unknown Material
Language:English
Published: eScholarship, University of California 2010
Subjects:
Online Access:https://escholarship.org/uc/item/82k1t32x
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spelling ftcdlib:oai:escholarship.org/ark:/13030/qt82k1t32x 2023-05-15T18:26:54+02:00 An investigation of the mechanical properties of the molten globule state of apomyoglobin Elms, Phillip James Marqusee, Susan 2010-01-01 application/pdf https://escholarship.org/uc/item/82k1t32x en eng eScholarship, University of California qt82k1t32x https://escholarship.org/uc/item/82k1t32x public Biophysics apomyoglobin force spectroscopy molten globule protein folding etd 2010 ftcdlib 2020-06-06T07:56:00Z Single molecule force spectroscopy has provided important insights into the properties and mechanisms of protein folding. However, there are still many unanswered questions about how force affects the folding and unfolding of proteins and, in particular, the relationship between force and the rate-limiting transition state. In this thesis, I developed two protein systems to address two specific questions. The first question arose form previous work on E. coli RNAse H, in which a molten globule-like intermediate was observed to have a large distance (5 ± 1 nm) to the transition state. This large distance was in sharp contrast to the smaller distances (< 2 nm) typically observed for natively folded proteins. This raised the question of whether this distance was a general property of the E. coli RNAse H intermediate or a more general property of a molten globule state. To this end, I investigated the equilibrium molten globule state of sperm whale apomyoglobin at pH 5 under force and demonstrated that this state had a large distance to the transition state of 6.1 ± 0.5 nm. Further, this state was shown to have a large distance to the transition state regardless of the axis of the applied force. This work suggests that a large distance to the transition state is a general property of the molten globule state. The second system was developed using the SH3 domain from chicken c-Src in order to investigate if and how the structure of the transition state changes under force. I investigated the behavior of the protein under two different force axes observing significant differences in the mechanical unfolding of the protein. These experiments are ongoing but indicate that the change in behavior is because of a change in the structure of the transition state under force. Finally, investigating the properties of the molten globule state revealed an error in previous methodology using constant force feedback experiments. In this thesis, I identify and explain the origin of this error. Further, work on the molten globule state required higher fidelity data and a more sophisticated approach for the analysis of the data. Working with John Chodera and colleagues, we implemented novel methods for the analysis of the data. Other/Unknown Material Sperm whale University of California: eScholarship
institution Open Polar
collection University of California: eScholarship
op_collection_id ftcdlib
language English
topic Biophysics
apomyoglobin
force spectroscopy
molten globule
protein folding
spellingShingle Biophysics
apomyoglobin
force spectroscopy
molten globule
protein folding
Elms, Phillip James
An investigation of the mechanical properties of the molten globule state of apomyoglobin
topic_facet Biophysics
apomyoglobin
force spectroscopy
molten globule
protein folding
description Single molecule force spectroscopy has provided important insights into the properties and mechanisms of protein folding. However, there are still many unanswered questions about how force affects the folding and unfolding of proteins and, in particular, the relationship between force and the rate-limiting transition state. In this thesis, I developed two protein systems to address two specific questions. The first question arose form previous work on E. coli RNAse H, in which a molten globule-like intermediate was observed to have a large distance (5 ± 1 nm) to the transition state. This large distance was in sharp contrast to the smaller distances (< 2 nm) typically observed for natively folded proteins. This raised the question of whether this distance was a general property of the E. coli RNAse H intermediate or a more general property of a molten globule state. To this end, I investigated the equilibrium molten globule state of sperm whale apomyoglobin at pH 5 under force and demonstrated that this state had a large distance to the transition state of 6.1 ± 0.5 nm. Further, this state was shown to have a large distance to the transition state regardless of the axis of the applied force. This work suggests that a large distance to the transition state is a general property of the molten globule state. The second system was developed using the SH3 domain from chicken c-Src in order to investigate if and how the structure of the transition state changes under force. I investigated the behavior of the protein under two different force axes observing significant differences in the mechanical unfolding of the protein. These experiments are ongoing but indicate that the change in behavior is because of a change in the structure of the transition state under force. Finally, investigating the properties of the molten globule state revealed an error in previous methodology using constant force feedback experiments. In this thesis, I identify and explain the origin of this error. Further, work on the molten globule state required higher fidelity data and a more sophisticated approach for the analysis of the data. Working with John Chodera and colleagues, we implemented novel methods for the analysis of the data.
author2 Marqusee, Susan
format Other/Unknown Material
author Elms, Phillip James
author_facet Elms, Phillip James
author_sort Elms, Phillip James
title An investigation of the mechanical properties of the molten globule state of apomyoglobin
title_short An investigation of the mechanical properties of the molten globule state of apomyoglobin
title_full An investigation of the mechanical properties of the molten globule state of apomyoglobin
title_fullStr An investigation of the mechanical properties of the molten globule state of apomyoglobin
title_full_unstemmed An investigation of the mechanical properties of the molten globule state of apomyoglobin
title_sort investigation of the mechanical properties of the molten globule state of apomyoglobin
publisher eScholarship, University of California
publishDate 2010
url https://escholarship.org/uc/item/82k1t32x
genre Sperm whale
genre_facet Sperm whale
op_relation qt82k1t32x
https://escholarship.org/uc/item/82k1t32x
op_rights public
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