SHEAR INDUCED DENATURATION OF PROTEINS

Lead Research Organisation: King's College London
Department Name: Engineering

Abstract

Proteins are fundamentally important molecules crucial for life and are now becoming widely used in industrial and medical applications. The protein drug industry alone is worth $US300billion per year and is growing quickly. Proteins are highly complex polymers and they have to fold into their correct structures to function effectively. This is not simple and the importance of protein folding has been long recognised and has led to decades of research into protein unfolding (or protein denaturation), with spin offs including medical applications (unfolded proteins can cause deadly diseases such as Alzheimer's and Parkinson's) and scientific technologies (protein unfolding tools are routinely used in biology and chemistry).Although protein unfolding by chemical and thermal means are areas of intensive research, and now mechanical unfolding is being utilised as a new tool in nanobioengineering, virtually nothing is known about unfolding by shear forces in fluids. Any new tool for controlling protein structure and unfolding will be a major breakthrough and the possibility of doing this using fluids (the natural environment for most proteins and most stages of protein preparation in industry), makes shear-flow an incredibly promising tool. However, we must discover the natural laws governing this phenomenon and develop the practical tools to measure and control shear-induced unfolding before we can make use of it.We will conduct the most comprehensive examination of the effects of shear flow on protein structure yet attempted, covering a diverse range of proteins of different shapes and stabilities, investigate the effects of experimental conditions and solvent properties, and use far more sensitive tools than have previously been brought to this problem. Our aim is to not only identify which proteins do or do not undergo unfolding under shear, but to identify and quantify which parts of the protein structure change (this is more physiologically important than just saying a protein does or doesn't unfold), learn the mechanisms of how shear-induced denaturation occurs and develop the methods to control and manipulate protein unfolding. This study will also involve the first comprehensive analysis of laminar and shear flow parameters in relation to proteins, which is required to obtain a true mechanistic understanding of the process.First, we will characterise and quantify the shear parameters of the flow cells to be used (both macro- and micro-fluidic devices) and then identify which proteins, from a widely varied set of targets, do or do not unfold in fluid flows. Different proteins can have greatly different structures and stabilities and it is likely that some proteins will not unfold under our experimental conditions, some will unfold, while others may need assistance to unfold by controlling experimental parameters (pH, viscosity etc.). It will be important to examine a number of very different proteins with different shapes and inherent stabilities to identify general trends or rules, and to identify favourable targets for the second phase of the project.We will then conduct more intensive studies for those proteins found to unfold under shear, with the aim of determining which parts of the protein structure change (which is more important than just knowing if the protein unfolds or not), quantifying these changes, detailing the mechanisms responsible and learning how to manipultae protein unfolding by controlling the solution characteristics (flow rate, viscosity, pH, chemical additives). In this way we will learn which types of proteins are most susceptible to shear flows and why, and we will develop the tools and techniques to control shear-induced denaturation, making it a new addition to the protein engineering toolkit.
 
Description Protein folding is one of the most fundamental problems in structural biology; protein misfolding and aggregation are closely linked with many debilitating diseases such as CJD and Alzheimer's. Scientists have employed various chemical or mechanical perturbation methods to study protein folding and unfolding. However, fluid flow or hydrodynamic shear has received little attention despite its important role in many biological processes (e.g. blood circulation). Protein molecules are exposed to shear both in their native environment but also during therapeutics and food production and thus shear induced unfolding is of both physiological and industrial importance. Prior to this joint project, it was unclear from the literature whether or not shear forces in fluids could perturb or denature protein structure. Using a novel interdisciplinary approach, we were the first group in UK to demonstrate that flow can indeed alter protein structure (Ashton et al. 2009). By combining Particle Image Velocimetry and Raman spectroscopy, we successfully monitored reversible, structural changes of proteins exposed to well controlled and characterised laminar flows (Dusting and Balabani, 2009; Imomoh et al, 2010) in situ. None of these changes would be detected post shearing.

The ability to monitor protein structure in situ in a fluid flow then allowed the importance of key structural parameters in stabilising a range of proteins against these flows to be investigated (Ashton et al. 2010). Experiments with a range of globular and less ordered proteins showed that proteins with increased size and beta-sheet content experienced less conformational changes. Thus, small proteins rich in a-helix, such as insulin, were found to be very susceptible to shear.

After addressing our original objectives we concentrated on determining whether fibrillation, the formation of amyloid-like fibrils, could be initiated or influenced by shear flows. Evidence in the literature suggests that flow promotes fibrillogenesis and affects fibril morphology but the physical mechanism involved is unclear. Incorporation of advanced chemometrics tools and other techniques (TEM imaging, FT-IR spectroscopy, fluorescence) with our methodology enabled the early onset (after 1 min of shearing) of fibril formation of insulin to be detected in situ under turbulent flow conditions (Webster et al 2011). Turbulent shear forces were found to be capable of promoting ordering of beta structure and lead to mature fibril formation, although enhanced mixing could also play a role. Experiments with other proteins and also using a rheometer indicated that although shear magnitude appears to be a more critical flow parameter than Reynolds number for promoting aggregation , the effects of fluid flow are more complex than originally thought. In an industrial or physiological environment proteins are not only subjected to shear forces of a certain magnitude but conditions that enhance mixing and interactions between proteins and interfaces. These might further promote conformational changes and fibrillogenesis and thus more effort is required to fully understand the role of fluid flow on protein structure.

Central to the success of this project has been the strong collaboration between our groups (Balabani: fluid dynamics; Blanch: bioRaman spectroscopy) with many visits by the post docs to their partner labs throughout the project. This has provided our post docs with extensive experience in fluid mechanics, spectroscopy and biophysics, a skillset that will be increasingly important as the potential of bioengineering proteins using fluid flows is used in the future to improve protein production yields and explore biophysical phenomena. It should be noted that this project follows on from our earlier Chemistry-Chemical Engineering Interface project (EP/D000696/1) whose aim was to establish a novel and lasting interdisciplinary collaboration, and so we have capitalised on that opportunity.
Exploitation Route Potential use in pharma/food industry, protein engineering and biotech companies. A significant number of outcomes were generated by the project clearly demonstrating impact and benefits to national economy. No exploitation or commercialisation has resulted from the project by us; however, it led to consultancy and further research and development by an SME company. Nevertheless, the research can be put into use through: a. further studies on the mechanical stability of proteins under shear, b. the development of computational tools that can accurately predict shear induced denaturation and c. the development of novel/sensitive tools for probing protein structure or for in situ screening of protein stability during food and manufacturing processes or drug delivery. Despite the originality and the success of the work we were unable to secure further funding from EPSRC to pursue these.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Research led to consultancy and subsequent involvement with an SME company; it led to subsequent funding and product development.
First Year Of Impact 2011
Sector Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Technology Strategy Board
Amount £95,997 (GBP)
Funding ID TSB100985 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 09/2011 
End 08/2013
 
Description KCL-Monash Exchange 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution During the project an exchange between the researcher on the project (J Dusting) and a researcher (J. Leontini) from Monash University, Australia took place to perform additional modelling work and develop further skills. This exchange also created a close link between the two groups.
 
Description MIB, University of Manchester 
Organisation University of Manchester
Department Manchester Institute of Biotechnology MIB
Country United Kingdom 
Sector Academic/University 
PI Contribution The project established a strong collaboration between the group of Dr Balabani and that of Dr Blanch at the MIB, University of Manchester.
 
Description Protein Technologies Ltd and Plymouth Marine Laboratories (PML) 
Organisation Plymouth Marine Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution The project and its disseminated activities helped in forming a close link with Protein Technologies Ltd initially through a consultancy and subsequently through further funding from TSB.
Collaborator Contribution Research and development, further funding; transfer of some of the findings to another application.
Impact Subsequent funding: TSB Technology Inspired Collaborate R& D, Voraxial Reactors in Large Scale Liquids Processing, Project 100985 (TP 4783-44 269), 2011-2013, 24 months duration, Total: £854,000, UCL: £97199 with Protein Technologies Ltd, Plymouth Marine Laboratory (PML), ETDE (PI) Closed September 2013 Publication:Simon F Thomas, Paul Rooks, Fabian Rudin, Neil Cagney, Stavroula Balabani, Sov Atkinson, Paul Goddard, Rachel Bransgrove, Paul T. Mason,Michael J. Allen. Swirl flow bioreactor containing dendritic copper-containing alginate beads: A potential rapid method for the eradication of Escherichia coli from waste water streams, J. Water Process Eng. (2014), http://dx.doi.org/10.1016/j.jwpe.2014.10.010
Start Year 2011
 
Description Protein Technologies Ltd and Plymouth Marine Laboratories (PML) 
Organisation Protein Technologies Ltd
Country United Kingdom 
Sector Private 
PI Contribution The project and its disseminated activities helped in forming a close link with Protein Technologies Ltd initially through a consultancy and subsequently through further funding from TSB.
 
Title microPIV facility 
Description The project provided an opportunity to enhance existing flow diagnostics equipment with the addition of an inverted microscope and sensitive camera to perform flow measurements in microscale flows. The facility has extended our research capabilities of the group; it provided us with access to the microfabrication facilities of LCN and enabled us to explore new collaborations and avenues of research eg by studying the behaviour of aggregating human blood in vitro. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2009 
Impact Significant research capability in imaging blood flows in vitro. 
 
Description Flow-induced conformational changes in proteins monitored in situ (ICAVS5, 2009). 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact ICAVS5, 5th International Conference on Advanced Vibrational Spectroscopy, Melbourne

Australia July 2009.
Year(s) Of Engagement Activity 2009
 
Description Flow-induced protein unfolding monitored using Raman Spectroscopy and Particle Image Velocimetry (UK) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Infrared Discussion group meeting, UK, Christmas 2009.
Year(s) Of Engagement Activity 2009
 
Description Probing protein mechanical stability with controlled shear flows (APS DFD conference, US) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at DFD09 Meeting of the American Physical Society, November 22-24, 2009, Minneapolis, Minnesota.
Year(s) Of Engagement Activity 2009
 
Description Protein structural changes occurring within flows. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 7th European Biophysics Congress, 11- 15 July, 2009, Genova, Italy.
Year(s) Of Engagement Activity 2009
 
Description Raman Spectroscopy and chemometrics to investigate time dependent physical changes of insulin in shear stress conditions (ICORS, 2010, Boston) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented at ICORS2010, Boston, US.
Year(s) Of Engagement Activity 2010
 
Description Raman spectroscopic studies of structural changes of insulin in controlled fluid flows (ICORS, Boston) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation at ICORS, 2010 Boston US.
Year(s) Of Engagement Activity 2010
 
Description Spatially and temporally-resolved measurement of laminar mixing induced by Taylor Vortex motion (Mixing Conference, London 2009) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 13th European Conference on Mixing, London, 14-17 April 2009.
Year(s) Of Engagement Activity 2009
 
Description Structures in Transitional Taylor-Couette flows identified using POD (APS DFD Meeting, 2009, US) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact DFD09 Meeting of the American Physical Society, November 22-24, 2009 Minneapolis, Minnesota.
Year(s) Of Engagement Activity 2009