Quaternary Structure and Dynamics of Polydisperse Molecular Chaperone Complexes
Lead Research Organisation:
University of Oxford
Department Name: Oxford Chemistry
Abstract
After their synthesis, the folding of proteins into their correct functional arrangements often requires assistance by 'molecular chaperones'. These proteins also play a vital role when the cell is stressed, by preserving and subsequently repairing damaged proteins. The mechanism by which these proteins carry out their 'housekeeping' duties is not fully understood, yet malfunctioning of this network results in a variety of disease states and even premature death. In this project we will focus on the least well understood family of the molecular chaperones, the Small Heat-Shock Proteins (sHSPs). In addition to their chaperone function, members of this protein family have been implicated in various disease states including cataract, motor neuropathy, and Alzheimer's disease.
Specifically, we will examine how sHSPs behave and interact with client proteins under stress conditions, and how they fit into the overall chaperone network in the cell. We have previously shown the sHSPs to be very dynamic, in that they can exchange subunits between each other on a rapid timescale. Their tendency to populate a range of states simultaneously, and to co-assemble with other sHSPs, has hampered conventional approaches to their study. We have focussed much of our previous research on developing and employing mass spectrometry (MS) strategies for the study of these proteins, and have developed a tool-kit well equipped for probing both their structure and dynamics.
The primary goal of this project is to examine in detail the complexes formed between the sHSPs and unfolding target proteins, which are the pivotal molecules in the sHSP chaperone pathway yet remain very poorly understood. We aim to characterise their organization, dynamic fluctuations, and their formation and disassembly. To achieve this ambitious target we will employ, and concomitantly develop further, a variety of MS-based approaches, including: the dissociation of the complexes in the gas phase; real-time reaction monitoring; and the direct measurement of their size.
To probe further the molecular details of these complexes between sHSP and target proteins we will perform molecular biology experiments in which we specifically mutate certain amino acids in the sHSPs to mimic modifications found in vivo. Thereby we can assess the effect of these alterations on the structure and dynamics of the complexes they form with substrate, allowing us to probe in detail the molecular mechanism of sHSP activity.
Therefore, through providing such insight into the way sHSPs interact with unfolding proteins we will go some way to understanding the pathway of their chaperone function. Furthermore, by clarifying their role in the overall molecular chaperone network, and examining the differences observed under conditions mimicking cellular stress, we hope to gain novel insight into the mechanism of their function.
Specifically, we will examine how sHSPs behave and interact with client proteins under stress conditions, and how they fit into the overall chaperone network in the cell. We have previously shown the sHSPs to be very dynamic, in that they can exchange subunits between each other on a rapid timescale. Their tendency to populate a range of states simultaneously, and to co-assemble with other sHSPs, has hampered conventional approaches to their study. We have focussed much of our previous research on developing and employing mass spectrometry (MS) strategies for the study of these proteins, and have developed a tool-kit well equipped for probing both their structure and dynamics.
The primary goal of this project is to examine in detail the complexes formed between the sHSPs and unfolding target proteins, which are the pivotal molecules in the sHSP chaperone pathway yet remain very poorly understood. We aim to characterise their organization, dynamic fluctuations, and their formation and disassembly. To achieve this ambitious target we will employ, and concomitantly develop further, a variety of MS-based approaches, including: the dissociation of the complexes in the gas phase; real-time reaction monitoring; and the direct measurement of their size.
To probe further the molecular details of these complexes between sHSP and target proteins we will perform molecular biology experiments in which we specifically mutate certain amino acids in the sHSPs to mimic modifications found in vivo. Thereby we can assess the effect of these alterations on the structure and dynamics of the complexes they form with substrate, allowing us to probe in detail the molecular mechanism of sHSP activity.
Therefore, through providing such insight into the way sHSPs interact with unfolding proteins we will go some way to understanding the pathway of their chaperone function. Furthermore, by clarifying their role in the overall molecular chaperone network, and examining the differences observed under conditions mimicking cellular stress, we hope to gain novel insight into the mechanism of their function.
Technical Summary
The Small Heat-Shock Proteins (sHSPs) are a family of molecular chaperones and crucial components of the protein homeostasis network. Despite their importance they remain poorly understood, both functionally and structurally, due largely to their tendency to populate a polydisperse ensemble of inter-converting oligomers at equilibrium.
These dynamics are integral to their chaperone function, and consequently to study the sHSPs structural biology techniques are required which have a high resolution of separation in both spatial and temporal dimensions. Mass spectrometry (MS) has over the last few years evolved to become an excellent approach for the investigation of protein assemblies, providing details as to their subunit architecture, equilibrium fluctuations, and transient interactions.
Since the establishment of my independent research group we have employed MS to investigate the structure and dynamics of sHSPs from a range of organisms. Here we propose to extend this work to capitalise on two recent and very exciting breakthroughs we have made. Firstly we have demonstrated that we can identify the individual oligomers which comprise the wide range of complexes formed between sHSPs and their targets. Secondly, we have developed a means for determining likely structures adopted by polydisperse sHSPs. We propose to combine these methodological developments to elucidate the quaternary architecture and dynamics of the extremely heterogeneous sHSP:target complexes.
Our strategy will employ, and further develop, cutting-edge MS-based methods for structural biology. We will investigate wild-type mammalian sHSPs, mutants mimicking post-translational modification, and variants known to display aberrant chaperone activity. This project will reveal how sHSPs interact with client proteins, thereby providing a clearer view of their function and their role in proteostasis.
These dynamics are integral to their chaperone function, and consequently to study the sHSPs structural biology techniques are required which have a high resolution of separation in both spatial and temporal dimensions. Mass spectrometry (MS) has over the last few years evolved to become an excellent approach for the investigation of protein assemblies, providing details as to their subunit architecture, equilibrium fluctuations, and transient interactions.
Since the establishment of my independent research group we have employed MS to investigate the structure and dynamics of sHSPs from a range of organisms. Here we propose to extend this work to capitalise on two recent and very exciting breakthroughs we have made. Firstly we have demonstrated that we can identify the individual oligomers which comprise the wide range of complexes formed between sHSPs and their targets. Secondly, we have developed a means for determining likely structures adopted by polydisperse sHSPs. We propose to combine these methodological developments to elucidate the quaternary architecture and dynamics of the extremely heterogeneous sHSP:target complexes.
Our strategy will employ, and further develop, cutting-edge MS-based methods for structural biology. We will investigate wild-type mammalian sHSPs, mutants mimicking post-translational modification, and variants known to display aberrant chaperone activity. This project will reveal how sHSPs interact with client proteins, thereby providing a clearer view of their function and their role in proteostasis.
Planned Impact
The proposed work will ultimately have practical importance within academia, industry, and have potential impact on human health. The research is directly applicable to four of BBSRC's strategic priority area: 'ageing research', 'systems approach to biological research', 'technology development for bioscience', and 'increased international collaboration'.
Ageing research: The protein homeostasis network of cell decreases in its efficiency during normal ageing, and can ultimately lead to a range of diseases. The sHSPs form a crucial component of this network, and therefore are candidates for pharmaceutical intervention. The work described here will provide insight into the pivotal complexes formed between sHSPs and their targets, and how they are regulated. This work will therefore be of immediate interest to companies and charities interested in the breakdown, and therapeutic re-establishment, of proteostasis.
Systems Approach to Biological Research: By providing insight into the structure and dynamics of the sHSPs and their interactions with targets, we will contribute of the 'systems view' of the cell. Similar to as described above, this will be important information for parties interested in how the cell maintains protein homeostasis. Furthermore there are interesting approaches to vaccine development being explored which rely on HSP:target complexes. Considering sHSPs are often major antigens (in M. tuberculosis for example), our characterisation of their interactions could potentially have considerable benefit for human health.
Technology Development for Bioscience: The technology and methodology we will employ, and further develop, in this project will be of wide applicability to structural biology in general. This is of direct interest to the MS manufacturers, whose market in the biosciences is continually expanding, and depend on breakthroughs into novel application areas.
Increased international collaboration: Our proposal includes collaboration with Prof Elizabeth Vierling at the University of Massachusetts, and the opportunity for the PDRA to visit her world-leading laboratories. This is in line with the BBSRC's encouragement of post-doctoral mobility and represents excellent training for the PDRA.
Ageing research: The protein homeostasis network of cell decreases in its efficiency during normal ageing, and can ultimately lead to a range of diseases. The sHSPs form a crucial component of this network, and therefore are candidates for pharmaceutical intervention. The work described here will provide insight into the pivotal complexes formed between sHSPs and their targets, and how they are regulated. This work will therefore be of immediate interest to companies and charities interested in the breakdown, and therapeutic re-establishment, of proteostasis.
Systems Approach to Biological Research: By providing insight into the structure and dynamics of the sHSPs and their interactions with targets, we will contribute of the 'systems view' of the cell. Similar to as described above, this will be important information for parties interested in how the cell maintains protein homeostasis. Furthermore there are interesting approaches to vaccine development being explored which rely on HSP:target complexes. Considering sHSPs are often major antigens (in M. tuberculosis for example), our characterisation of their interactions could potentially have considerable benefit for human health.
Technology Development for Bioscience: The technology and methodology we will employ, and further develop, in this project will be of wide applicability to structural biology in general. This is of direct interest to the MS manufacturers, whose market in the biosciences is continually expanding, and depend on breakthroughs into novel application areas.
Increased international collaboration: Our proposal includes collaboration with Prof Elizabeth Vierling at the University of Massachusetts, and the opportunity for the PDRA to visit her world-leading laboratories. This is in line with the BBSRC's encouragement of post-doctoral mobility and represents excellent training for the PDRA.
People |
ORCID iD |
Justin Benesch (Principal Investigator) |
Publications
Alderson T
(2018)
Local unfolding of the HSP27 monomer regulates chaperone activity
Alderson T
(2020)
Conditional Disorder in Small Heat-shock Proteins
in Journal of Molecular Biology
Alderson TR
(2017)
Proline isomerization in the C-terminal region of HSP27.
in Cell stress & chaperones
Alderson TR
(2019)
Local unfolding of the HSP27 monomer regulates chaperone activity.
in Nature communications
Aprile FA
(2013)
Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.
in PloS one
Baldwin AJ
(2012)
Probing dynamic conformations of the high-molecular-weight aB-crystallin heat shock protein ensemble by NMR spectroscopy.
in Journal of the American Chemical Society
Benesch J
(2014)
Native Mass Spectrometry for Structural Biophysics
in Biophysical Journal
Clark AR
(2018)
Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3.
in Journal of molecular biology
Collier M
(2019)
HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C
in Science Advances
Hilton GR
(2013)
C-terminal interactions mediate the quaternary dynamics of aB-crystallin.
in Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Hilton GR
(2012)
Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry.
in Journal of the Royal Society, Interface
Hochberg G
(2015)
The Big Book on Small Heat Shock Proteins
Hochberg GK
(2014)
The structured core domain of aB-crystallin can prevent amyloid fibrillation and associated toxicity.
in Proceedings of the National Academy of Sciences of the United States of America
Hochberg GK
(2014)
Dynamical structure of aB-crystallin.
in Progress in biophysics and molecular biology
Hochberg GKA
(2018)
Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions.
in Science (New York, N.Y.)
Jovcevski B
(2018)
The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of aB-crystallin.
in Cell stress & chaperones
Jovcevski B
(2015)
Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.
in Chemistry & biology
Jovcevski B
(2017)
Evaluating the Effect of Phosphorylation on the Structure and Dynamics of Hsp27 Dimers by Means of Ion Mobility Mass Spectrometry.
in Analytical chemistry
Kondrat FD
(2015)
Native mass spectrometry: towards high-throughput structural proteomics.
in Methods in molecular biology (Clifton, N.J.)
Lyon YA
(2019)
Structural and functional consequences of age-related isomerization in a-crystallins.
in The Journal of biological chemistry
Marklund EG
(2018)
Structural and functional aspects of the interaction partners of the small heat-shock protein in Synechocystis.
in Cell stress & chaperones
Mitzelfelt KA
(2016)
The Human 343delT HSPB5 Chaperone Associated with Early-onset Skeletal Myopathy Causes Defects in Protein Solubility.
in The Journal of biological chemistry
Reid Alderson T
(2021)
A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins
in The EMBO Journal
Santhanagopalan I
(2018)
It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate.
in The Journal of biological chemistry
Stengel F
(2012)
Dissecting Heterogeneous Molecular Chaperone Complexes Using a Mass Spectrum Deconvolution Approach
in Chemistry & Biology
Description | Improved understanding of how the cell copes with stress, in particular the involvement of "molecular chaperone" proteins. |
Exploitation Route | To further understand how the cell copes with stress: with benefit to acute (e.g. heat stroke), chronic (e.g. ageing) conditions. Also relevant to plants and their resistance to abiotic stresses (e.g. climate change leading to higher temperature) |
Sectors | Agriculture Food and Drink Pharmaceuticals and Medical Biotechnology |
Description | Continued collaboration with the mass spectrometry industry, and underpinning science that led to our spinout Refeyn. |
First Year Of Impact | 2015 |
Sector | Pharmaceuticals and Medical Biotechnology |
Description | GCRF |
Amount | £43,966 (GBP) |
Organisation | Higher Education Funding Council for England |
Sector | Public |
Country | United Kingdom |
Start | 03/2018 |
End | 07/2018 |
Description | Ecroyd lab |
Organisation | University of Wollongong |
Country | Australia |
Sector | Academic/University |
PI Contribution | Long-term collaboration - exchange of expertise, reagents, and co-authorship of papers |
Collaborator Contribution | Long-term collaboration - exchange of expertise, reagents, and co-authorship of papers |
Impact | See publications |
Start Year | 2010 |
Description | Julian lab |
Organisation | University of California, Riverside |
Country | United States |
Sector | Academic/University |
PI Contribution | Collaboration: exchange of expertise, reagents, and co-authorship of publications |
Collaborator Contribution | Collaboration: exchange of expertise, reagents, and co-authorship of publications |
Impact | See publications |
Start Year | 2017 |
Description | McHaourab lab |
Organisation | Vanderbilt University |
Country | United States |
Sector | Academic/University |
PI Contribution | Collaboration - exchange of expertise, reagents, and co-authorship of papers |
Collaborator Contribution | Collaboration - exchange of expertise, reagents, and co-authorship of papers |
Impact | See publications |
Start Year | 2016 |
Description | Vierling lab |
Organisation | University of Massachusetts |
Country | United States |
Sector | Academic/University |
PI Contribution | Long-term collaboration - exchange of expertise and reagents, and co-authorship |
Collaborator Contribution | Long-term collaboration - exchange of expertise and reagents, and co-authorship |
Impact | See publications |
Company Name | Refeyn |
Description | Refeyn develops technology that is capable of measuring and imaging molecular mass. |
Year Established | 2018 |
Impact | Refeyn and sold and delivered instruments to labs around the world, with researchers in academia and industry using the instrumentation to perform new science. The company is revenue generating, and has raised significant amount of venture capital funding, and has created a diverse set of jobs. |
Website | http://www.refeyn.com |
Description | Bratislava Childrens University |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Outreach presentation to school children from across Slovakia |
Year(s) Of Engagement Activity | 2014 |
Description | MPLS blog - paralog Science paper |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Press release in blog format regarding high profile paper |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.mpls.ox.ac.uk/news/proteins-assemble-study-sheds-new-light-on-our-biochemical-workhorses |
Description | School visit (Montessori) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Outreach talk to school children aged 8-13 |
Year(s) Of Engagement Activity | 2015 |
Description | |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Media (as a channel to the public) |
Results and Impact | Twitter account highlight research and related areas of interest |
Year(s) Of Engagement Activity | 2012 |
URL | https://twitter.com/beneschresearch |