Christopher M. Dobson

Sequential Release of Proteins from Structured Multishell Microcapsules

Co-reporter:Ulyana Shimanovich, Thomas C. T. Michaels, Erwin De Genst, Dijana Matak-Vinkovic, Christopher M. Dobson, and Tuomas P. J. Knowles

Biomacromolecules October 9, 2017 Volume 18(Issue 10) pp:3052-3052

Publication Date(Web):August 9, 2017

DOI:10.1021/acs.biomac.7b00351

In nature, a wide range of functional materials is based on proteins. Increasing attention is also turning to the use of proteins as artificial biomaterials in the form of films, gels, particles, and fibrils that offer great potential for applications in areas ranging from molecular medicine to materials science. To date, however, most such applications have been limited to single component materials despite the fact that their natural analogues are composed of multiple types of proteins with a variety of functionalities that are coassembled in a highly organized manner on the micrometer scale, a process that is currently challenging to achieve in the laboratory. Here, we demonstrate the fabrication of multicomponent protein microcapsules where the different components are positioned in a controlled manner. We use molecular self-assembly to generate multicomponent structures on the nanometer scale and droplet microfluidics to bring together the different components on the micrometer scale. Using this approach, we synthesize a wide range of multiprotein microcapsules containing three well-characterized proteins: glucagon, insulin, and lysozyme. The localization of each protein component in multishell microcapsules has been detected by labeling protein molecules with different fluorophores, and the final three-dimensional microcapsule structure has been resolved by using confocal microscopy together with image analysis techniques. In addition, we show that these structures can be used to tailor the release of such functional proteins in a sequential manner. Moreover, our observations demonstrate that the protein release mechanism from multishell capsules is driven by the kinetic control of mass transport of the cargo and by the dissolution of the shells. The ability to generate artificial materials that incorporate a variety of different proteins with distinct functionalities increases the breadth of the potential applications of artificial protein-based materials and provides opportunities to design more refined functional protein delivery systems.

Hierarchical Biomolecular Emulsions Using 3-D Microfluidics with Uniform Surface Chemistry

Co-reporter:Zenon Toprakcioglu, Aviad Levin, and Tuomas P. J. Knowles

Biomacromolecules November 13, 2017 Volume 18(Issue 11) pp:3642-3642

Publication Date(Web):September 29, 2017

DOI:10.1021/acs.biomac.7b01159

Microfluidic devices can be used to produce single, double and higher order emulsions, where droplet sizes can be precisely controlled and modulated. Such emulsions have great potential for the storage and study of biomolecules, including peptides and proteins. However, advancement of this technique has remained challenging due to the tendency of various biomolecules to adhere to the surface of the formed channels, resulting in changes in surface wetting and fouling on the micrometer scale. Thus, precise control of surface wettability plays a crucial role in the processes that govern droplet formation. Here, we report an approach for producing both water–oil–water (w/o/w) and oil–water–oil (o/w/o) double emulsions without any need for surface modification, an enabling feature for biomolecular encapsulation. Using this strategy, we show that the number of monodisperse encapsulated internal droplets can be controlled systematically and reproducibly by suitable adjustment of the relevant flow rates, and ranges from 1 to 40 in the case of w/o/w emulsions. We further demonstrate that the number of internal droplets scales linearly with the reciprocal flow rate of the outer continuous phase, when the inner and middle phase flow rates are kept constant. We demonstrate that this approach is suitable for forming double emulsions where the inner phase consists of reconstituted silk protein solution whereby incubation of the internal droplets can be induced to form a gel resulting in silk fibroin microgels surrounded by an external oil shell. Finally, for o/w/o emulsions, we show that single or multiple monodisperse internal droplets can be encapsulated with a size that ranges over 1 order of magnitude, from ca. 10 μm to >100 μm. Moreover, o/w/o emulsions where the middle phase consists of silk fibroin solution were prepared and by allowing the protein to aggregate, a core–shell structure was formed. This microfluidic strategy allows for multiple emulsions to be generated drop by drop for biomolecular solutions with potential applications in the biomedical and pharmaceutical fields.

Scaling behaviour and rate-determining steps in filamentous self-assembly

Co-reporter:Georg Meisl;Luke Rajah;Samuel A. I. Cohen;Manuela Pfammatter;Anđela Šarić;Erik Hellstrand;Alexander K. Buell;Adriano Aguzzi;Sara Linse;Michele Vendruscolo;Tuomas P. J. Knowles

Chemical Science (2010-Present) 2017 vol. 8(Issue 10) pp:7087-7097

Publication Date(Web):2017/09/25

DOI:10.1039/C7SC01965C

The formation of filaments from naturally occurring protein molecules is a process at the core of a range of functional and aberrant biological phenomena, such as the assembly of the cytoskeleton or the appearance of aggregates in Alzheimer's disease. The macroscopic behaviour associated with such processes is remarkably diverse, ranging from simple nucleated growth to highly cooperative processes with a well-defined lagtime. Thus, conventionally, different molecular mechanisms have been used to explain the self-assembly of different proteins. Here we show that this range of behaviour can be quantitatively captured by a single unifying Petri net that describes filamentous growth in terms of aggregate number and aggregate mass concentrations. By considering general features associated with a particular network connectivity, we are able to establish directly the rate-determining steps of the overall aggregation reaction from the system's scaling behaviour. We illustrate the power of this framework on a range of different experimental and simulated aggregating systems. The approach is general and will be applicable to any future extensions of the reaction network of filamentous self-assembly.

Nanoscale click-reactive scaffolds from peptide self-assembly

Co-reporter:Alexander P. M. Guttenplan;Laurence J. Young

Journal of Nanobiotechnology 2017 Volume 15( Issue 1) pp:

Publication Date(Web):

DOI:10.1186/s12951-017-0300-7

Nanoscale click-reactive scaffolds from peptide self-assembly

Co-reporter:Alexander P. M. Guttenplan;Laurence J. Young

Journal of Nanobiotechnology 2017 Volume 15( Issue 1) pp:

Publication Date(Web):

DOI:10.1186/s12951-017-0300-7

Gradient-free determination of isoelectric points of proteins on chip

Co-reporter:Urszula Łapińska;Kadi L. Saar;Emma V. Yates;Therese W. Herling;Thomas Müller;Pavan K. Challa;Tuomas P. J. Knowles

Physical Chemistry Chemical Physics 2017 vol. 19(Issue 34) pp:23060-23067

Publication Date(Web):2017/08/30

DOI:10.1039/C7CP01503H

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.

Modulation of electrostatic interactions to reveal a reaction network unifying the aggregation behaviour of the Aβ42 peptide and its variants

Co-reporter:Georg Meisl;Xiaoting Yang;Sara Linse;Tuomas P. J. Knowles

Chemical Science (2010-Present) 2017 vol. 8(Issue 6) pp:4352-4362

Publication Date(Web):2017/05/30

DOI:10.1039/C7SC00215G

The aggregation of the amyloid β peptide (Aβ42), which is linked to Alzheimer's disease, can be altered significantly by modulations of the peptide's intermolecular electrostatic interactions. Variations in sequence and solution conditions have been found to lead to highly variable aggregation behaviour. Here we modulate systematically the electrostatic interactions governing the aggregation kinetics by varying the ionic strength of the solution. We find that changes in the solution ionic strength induce a switch in the reaction pathway, altering the dominant mechanisms of aggregate multiplication. This strategy thereby allows us to continuously sample a large space of different reaction mechanisms and develop a minimal reaction network that unifies the experimental kinetics under a wide range of different conditions. More generally, this universal reaction network connects previously separate systems, such as charge mutants of the Aβ42 peptide, on a continuous mechanistic landscape, providing a unified picture of the aggregation mechanism of Aβ42.

Nanoscale click-reactive scaffolds from peptide self-assembly

Co-reporter:Alexander P. M. Guttenplan;Laurence J. Young

Journal of Nanobiotechnology 2017 Volume 15( Issue 1) pp:70

Publication Date(Web):06 October 2017

DOI:10.1186/s12951-017-0300-7

Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled.Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of “click” reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology.Although previous studies have produced peptides that can both form amyloid fibrils and undergo “click”-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.

Nanoscale click-reactive scaffolds from peptide self-assembly

Co-reporter:Alexander P. M. Guttenplan;Laurence J. Young

Journal of Nanobiotechnology 2017 Volume 15( Issue 1) pp:70

Publication Date(Web):06 October 2017

DOI:10.1186/s12951-017-0300-7

Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled.Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of “click” reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology.Although previous studies have produced peptides that can both form amyloid fibrils and undergo “click”-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer’s disease

Co-reporter:Johnny Habchi;Oskar Hansson;Sean Chia;Janet R. Kumita;Samuel I. A. Cohen;Michele Perni;Pavan Kumar Challa;Tuomas P. J. Knowles;Paolo Arosio;Michele Vendruscolo;Minkoo Ahn;Ryan Limbocker;Benedetta Mannini;Sara Linse

PNAS 2017 Volume 114 (Issue 2 ) pp:E200-E208

Publication Date(Web):2017-01-10

DOI:10.1073/pnas.1615613114

The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer’s disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a read-out the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.

Amyloid Fibrils as Building Blocks for Natural and Artificial Functional Materials

Co-reporter:Tuomas P. J. Knowles;Raffaele Mezzenga

Advanced Materials 2016 Volume 28( Issue 31) pp:6546-6561

Publication Date(Web):

DOI:10.1002/adma.201505961

Proteinaceous materials based on the amyloid core structure have recently been discovered at the origin of biological functionality in a remarkably diverse set of roles, and attention is increasingly turning towards such structures as the basis of artificial self-assembling materials. These roles contrast markedly with the original picture of amyloid fibrils as inherently pathological structures. Here we outline the salient features of this class of functional materials, both in the context of the functional roles that have been revealed for amyloid fibrils in nature, as well as in relation to their potential as artificial materials. We discuss how amyloid materials exemplify the emergence of function from protein self-assembly at multiple length scales. We focus on the connections between mesoscale structure and material function, and demonstrate how the natural examples of functional amyloids illuminate the potential applications for future artificial protein based materials.

Synthesis of Nonequilibrium Supramolecular Peptide Polymers on a Microfluidic Platform

Co-reporter:Thomas O. Mason; Thomas C. T. Michaels; Aviad Levin; Ehud Gazit; Christopher M. Dobson; Alexander K. Buell;Tuomas P. J. Knowles

Journal of the American Chemical Society 2016 Volume 138(Issue 30) pp:9589-9596

Publication Date(Web):July 7, 2016

DOI:10.1021/jacs.6b04136

The self-assembly of peptides and peptide mimetics into supramolecular polymers has been established in recent years as a route to biocompatible nanomaterials with novel mechanical, optical, and electronic properties. The morphologies of the resulting polymers are usually dictated by the strengths as well as lifetimes of the noncovalent bonds that lead to the formation of the structures. Together with an often incomplete understanding of the assembly mechanisms, these factors limit the control over the formation of polymers with tailored structures. Here, we have developed a microfluidic flow reactor to measure growth rates directly and accurately on the axial and radial faces of crystalline peptide supramolecular polymers. We show that the structures grow through two-dimensional nucleation mechanisms, with rates that depend exponentially on the concentration of soluble peptide. Using these mechanistic insights into the growth behavior of the axial and radial faces, we have been able to tune the aspect ratio of populations of dipeptide assemblies. These results demonstrate a general strategy to control kinetically self-assembly beyond thermodynamic products governed by the intrinsic properties of the building blocks in order to attain the required morphology and function.

Microfluidic Diffusion Viscometer for Rapid Analysis of Complex Solutions

Co-reporter:Paolo Arosio, Kevin Hu, Francesco A. Aprile, Thomas Müller, and Tuomas P. J. Knowles

Analytical Chemistry 2016 Volume 88(Issue 7) pp:3488

Publication Date(Web):March 4, 2016

DOI:10.1021/acs.analchem.5b02930

The viscosity of complex solutions is a physical property of central relevance for a large number of applications in material, biological, and biotechnological sciences. Here we demonstrate a microfluidic technology to measure the viscosity of solutions by following the advection and diffusion of tracer particles under steady-state flow. We validate our method with standard water-glycerol mixtures, and then we apply this microfluidic diffusion viscometer to measure the viscosity of protein solutions at high concentrations as well as of a crude cell lysate. Our approach exhibits a series of attractive features, including analysis time on the order of seconds and the consumption of a few μL of sample, as well as the possibility to readily integrate the microfluidic viscometer in other instrument platforms or modular microfluidic devices. These characteristics make microfluidic diffusion viscometry an attractive approach in automated processes in biotechnology and health-care sciences where fast measurements with limited amount of sample consumption are required.

Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation

Co-reporter:Paolo Arosio, Tommy Cedervall, Tuomas P.J. Knowles, Sara Linse

Analytical Biochemistry 2016 Volume 504() pp:7-13

Publication Date(Web):1 July 2016

DOI:10.1016/j.ab.2016.03.015

Abstract

The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1–42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril–fibril assembly can play in the deposition of aggregation-prone peptides.

Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions

Co-reporter:Paolo Arosio, Thomas Müller, Luke Rajah, Emma V. Yates, Francesco A. Aprile, Yingbo Zhang, Samuel I. A. Cohen, Duncan A. White, Therese W. Herling, Erwin J. De Genst, Sara Linse, Michele Vendruscolo, Christopher M. Dobson, and Tuomas P. J. Knowles

ACS Nano 2016 Volume 10(Issue 1) pp:333

Publication Date(Web):December 17, 2015

DOI:10.1021/acsnano.5b04713

Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space–time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.Keywords: diffusion; immunoassay; interactions; polydispersity; proteins; size distribution;

An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer’s disease

Co-reporter:Martin B. D. Müller;Ana Rita Costa;Maho Yagi-Utsumi;Priyanka Joshi;Johnny Habchi;Sara Linse;Samuel I. A. Cohen;Tuomas P. J. Knowles;Paolo Arosio;Ellen A. A. Nollen;Michele Vendruscolo;Michele Perni;Sean Chia

Science Advances 2016 Volume 2(Issue 2) pp:e1501244

Publication Date(Web):12 Feb 2016

DOI:10.1126/sciadv.1501244

An approved anticancer drug selectively targets the first step in the molecular cascade resulting in Alzheimer’s disease.

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading

Co-reporter:Marija Iljina;Gonzalo A. Garcia;Mathew H. Horrocks;Laura Tosatto;Minee L. Choi;Kristina A. Ganzinger;Andrey Y. Abramov;Sonia Gandhi;Nicholas W. Wood;Nunilo Cremades;Tuomas P. J. Knowles;David Klenerman;

Proceedings of the National Academy of Sciences 2016 113(9) pp:E1206-E1215

Publication Date(Web):February 16, 2016

DOI:10.1073/pnas.1524128113

The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson’s disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5–140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10⁴, are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

An Environmentally Sensitive Fluorescent Dye as a Multidimensional Probe of Amyloid Formation

Co-reporter:Emma V. Yates, Georg Meisl, Tuomas P. J. Knowles, and Christopher M. Dobson

The Journal of Physical Chemistry B 2016 Volume 120(Issue 9) pp:2087-2094

Publication Date(Web):February 11, 2016

DOI:10.1021/acs.jpcb.5b09663

We have explored amyloid formation using poly(amino acid) model systems in which differences in peptide secondary structure and hydrophobicity can be introduced in a controlled manner. We show that an environmentally sensitive fluorescent dye, dapoxyl, is able to identify β-sheet structure and hydrophobic surfaces, structural features likely to be related to toxicity, as a result of changes in its excitation and emission profiles and its relative quantum yield. These results show that dapoxyl is a multidimensional probe of the time dependence of amyloid aggregation, which provides information about the presence and nature of metastable aggregation intermediates that is inaccessible to the conventional probes that rely on changes in quantum yield alone.

A High Power-Density, Mediator-Free, Microfluidic Biophotovoltaic Device for Cyanobacterial Cells

Co-reporter:Paolo Bombelli;Thomas Müller;Therese W. Herling;Christopher J. Howe;Tuomas P. J. Knowles

Advanced Energy Materials 2015 Volume 5( Issue 2) pp:

Publication Date(Web):

DOI:10.1002/aenm.201401299

Biophotovoltaics has emerged as a promising technology for generating renewable energy because it relies on living organisms as inexpensive, self-repairing, and readily available catalysts to produce electricity from an abundant resource: sunlight. The efficiency of biophotovoltaic cells, however, has remained significantly lower than that achievable through synthetic materials. Here, a platform is devised to harness the large power densities afforded by miniaturized geometries. To this effect, a soft-lithography approach is developed for the fabrication of microfluidic biophotovoltaic devices that do not require membranes or mediators. Synechocystis sp. PCC 6803 cells are injected and allowed to settle on the anode, permitting the physical proximity between cells and electrode required for mediator-free operation. Power densities of above 100 mW m^-2 are demonstrated for a chlorophyll concentration of 100 μM under white light, which is a high value for biophotovoltaic devices without extrinsic supply of additional energy.

Biophysical approaches for the study of interactions between molecular chaperones and protein aggregates

Co-reporter:Maya A. Wright, Francesco A. Aprile, Paolo Arosio, Michele Vendruscolo, Christopher M. Dobson and Tuomas P. J. Knowles

Chemical Communications 2015 vol. 51(Issue 77) pp:14425-14434

Publication Date(Web):10 Aug 2015

DOI:10.1039/C5CC03689E

Molecular chaperones are key components of the arsenal of cellular defence mechanisms active against protein aggregation. In addition to their established role in assisting protein folding, increasing evidence indicates that molecular chaperones are able to protect against a range of potentially damaging aspects of protein behaviour, including misfolding and aggregation events that can result in the generation of aberrant protein assemblies whose formation is implicated in the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The interactions between molecular chaperones and different amyloidogenic protein species are difficult to study owing to the inherent heterogeneity of the aggregation process as well as the dynamic nature of molecular chaperones under physiological conditions. As a consequence, understanding the detailed microscopic mechanisms underlying the nature and means of inhibition of aggregate formation remains challenging yet is a key objective for protein biophysics. In this review, we discuss recent results from biophysical studies on the interactions between molecular chaperones and protein aggregates. In particular, we focus on the insights gained from current experimental techniques into the dynamics of the oligomerisation process of molecular chaperones, and highlight the opportunities that future biophysical approaches have in advancing our understanding of the great variety of biological functions of this important class of proteins.

A microfluidic platform for quantitative measurements of effective protein charges and single ion binding in solution

Co-reporter:Therese W. Herling, Paolo Arosio, Thomas Müller, Sara Linse and Tuomas P. J. Knowles

Physical Chemistry Chemical Physics 2015 vol. 17(Issue 18) pp:12161-12167

Publication Date(Web):16 Apr 2015

DOI:10.1039/C5CP00746A

The charge state of proteins in solution is a key biophysical parameter that modulates both long and short range macromolecular interactions. However, unlike in the case of many small molecules, the effective charges of complex biomolecules in solution cannot in general be predicted reliably from their chemical structures alone. Here we present an approach for quantifying the effective charges of solvated biomolecules from independent measurements of their electrophoretic mobilities and diffusion coefficients in free solution within a microfluidic device. We illustrate the potential of this approach by determining the effective charges of a charge-ladder family of mutants of the calcium binding protein calbindin D9k in solution under native conditions. Furthermore, we explore ion-binding under native conditions, and demonstrate the ability to detect the chelation of a single calcium ion through the change that ion binding imparts on the effective charge of calbindin D9k. Our findings highlight the difference between the dry sequence charge and the effective charge of proteins in solution, and open up a route towards rapid and quantitative charge measurements in small volumes in the condensed phase.

Multiphase Protein Microgels

Co-reporter:Ulyana Shimanovich;Yang Song;Jasna Brujic;Ho Cheung Shum;Tuomas P. J. Knowles

Macromolecular Bioscience 2015 Volume 15( Issue 4) pp:501-508

Publication Date(Web):

DOI:10.1002/mabi.201400366

Peptides and proteins represent attractive building blocks for the development of new functional materials due to the biocompatibility and biodegradability of many naturally abundant proteins. In nature, sophisticated material functionality is commonly achieved through spatial control of protein localisation and structure on both the nano and micro scales. We approached this requirement in an artificial setting by exploiting the propensity of proteins to self-assemble into amyloid fibrils to achieve nano scale order, and utilised aqueous liquid/liquid phase separation to control the micron scale localization of the proteinaceous component under microconfinement. We show that in combination with droplet microfluidics, this strategy allows the synthesis of core-shell microgel particles composed of protein nanofibrils.

Molecular Rotors Provide Insights into Microscopic Structural Changes During Protein Aggregation

Co-reporter:Alexander J. Thompson, Therese W. Herling, Markéta Kubánková, Aurimas Vyšniauskas, Tuomas P. J. Knowles, and Marina K. Kuimova

The Journal of Physical Chemistry B 2015 Volume 119(Issue 32) pp:10170-10179

Publication Date(Web):July 20, 2015

DOI:10.1021/acs.jpcb.5b05099

Changes in microscopic viscosity represent an important characteristic of structural transitions in soft matter systems. Here we demonstrate the use of molecular rotors to explore the changes in microrheology accompanying the transition of proteins from their soluble states into a gel phase composed of amyloid fibrils. The formation of beta-sheet rich protein aggregates, including amyloid fibrils, is a hallmark of a number of neurodegenerative disorders, and as such, the mechanistic details of this process are actively sought after. In our experiments, molecular rotors report an increase in rigidity of approximately three orders of magnitude during the aggregation reaction. Moreover, phasor analysis of the fluorescence decay signal from the molecular rotors suggests the presence of multiple distinct mechanistic stages during the aggregation process. Our results show that molecular rotors can reveal key microrheological features of protein systems not observable through classical fluorescent probes operating in light switch mode.

Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry

Co-reporter:Xiao-Ming Zhou, Ulyana Shimanovich, Therese W. Herling, Si Wu, Christopher M. Dobson, Tuomas P. J. Knowles, and Sarah Perrett

ACS Nano 2015 Volume 9(Issue 6) pp:5772

Publication Date(Web):June 1, 2015

DOI:10.1021/acsnano.5b00061

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions.Keywords: alkaline phosphatase; amyloid fibrils; enzymatic microgel; microfluidics; Ure2;

Force generation by the growth of amyloid aggregates

Co-reporter:Therese W. Herling;Gonzalo A. Garcia;Wolfgang Grentz;James Dean;Thomas C. T. Michaels;Ulyana Shimanovich;Hongze Gang;Eugene M. Terentjev;Thomas Müller;Batuhan Kav;Tuomas P. J. Knowles

PNAS 2015 Volume 112 (Issue 31 ) pp:9524-9529

Publication Date(Web):2015-08-04

DOI:10.1073/pnas.1417326112

The generation of mechanical forces are central to a wide range of vital biological processes, including the function of the cytoskeleton. Although the forces emerging from the polymerization of native proteins have been studied in detail, the potential for force generation by aberrant protein polymerization has not yet been explored. Here, we show that the growth of amyloid fibrils, archetypical aberrant protein polymers, is capable of unleashing mechanical forces on the piconewton scale for individual filaments. We apply microfluidic techniques to measure the forces released by amyloid growth for two systems: insulin and lysozyme. The level of force measured for amyloid growth in both systems is comparable to that observed for actin and tubulin, systems that have evolved to generate force during their native functions and, unlike amyloid growth, rely on the input of external energy in the form of nucleotide hydrolysis for maximum force generation. Furthermore, we find that the power density released from growing amyloid fibrils is comparable to that of high-performance synthetic polymer actuators. These findings highlight the potential of amyloid structures as active materials and shed light on the criteria for regulation and reversibility that guide molecular evolution of functional polymers.

Protein Microgels from Amyloid Fibril Networks

Co-reporter:Ulyana Shimanovich, Igor Efimov, Thomas O. Mason, Patrick Flagmeier, Alexander K. Buell, Aharon Gedanken, Sara Linse, Karin S. Åkerfeldt, Christopher M. Dobson, David A. Weitz, and Tuomas P. J. Knowles

ACS Nano 2015 Volume 9(Issue 1) pp:43

Publication Date(Web):December 3, 2014

DOI:10.1021/nn504869d

Nanofibrillar forms of proteins were initially recognized in the context of pathology, but more recently have been discovered in a range of functional roles in nature, including as active catalytic scaffolds and bacterial coatings. Here we show that protein nanofibrils can be used to form the basis of monodisperse microgels and gel shells composed of naturally occurring proteins. We explore the potential of these protein microgels to act as drug carrier agents, and demonstrate the controlled release of four different encapsulated drug-like small molecules, as well as the component proteins themselves. Furthermore, we show that protein nanofibril self-assembly can continue after the initial formation of the microgel particles, and that this process results in active materials with network densities that can be modulated in situ. We demonstrate that these materials are nontoxic to human cells and that they can be used to enhance the efficacy of antibiotics relative to delivery in homogeneous solution. Because of the biocompatibility and biodegradability of natural proteins used in the fabrication of the microgels, as well as their ability to control the release of small molecules and biopolymers, protein nanofibril microgels represent a promising class of functional artificial multiscale materials generated from natural building blocks.Keywords: drug release; lysozyme; microfluidics; microgels; protein nanofibrils;

Preventing peptide and protein misbehavior

Co-reporter:Paolo Arosio;Georg Meisl;Tuomas P. J. Knowles;Maria Andreasen

PNAS 2015 Volume 112 (Issue 17 ) pp:5267-5268

Publication Date(Web):2015-04-28

DOI:10.1073/pnas.1505170112

Density-Gradient-Free Microfluidic Centrifugation for Analytical and Preparative Separation of Nanoparticles

Co-reporter:Paolo Arosio, Thomas Müller, L. Mahadevan, and Tuomas P. J. Knowles

Nano Letters 2014 Volume 14(Issue 5) pp:2365-2371

Publication Date(Web):March 10, 2014

DOI:10.1021/nl404771g

Sedimentation and centrifugation techniques are widely applied for the separation of biomolecules and colloids but require the presence of controlled density gradients for stable operation. Here we present an approach for separating nanoparticles in free solution without gradients. We use microfluidics to generate a convective flow perpendicular to the sedimentation direction. We show that the hydrodynamic Rayleigh–Taylor-like instability, which, in traditional methods, requires the presence of a density gradient, can be suppressed by the Poiseuille flow in the microchannel. We illustrate the power of this approach by demonstrating the separation of mixtures of particles on the nanometer scale, orders of magnitude smaller than the micrometer-sized objects separated by conventional inertial microfluidic approaches. This technique exhibits a series of favorable features including short analysis time, small sample volume, limited dilution of the analyte, limited interactions with surfaces as well as the possibility to tune easily the separation range by adjusting the geometry of the system. These features highlight the potential of gradient-free microfluidic centrifugation as an attractive route toward a broad range of nanoscale applications.

Diffuse transition state structure for the unfolding of a leucine-rich repeat protein

Co-reporter:Sadie E. Kelly, Georg Meisl, Pamela J. E. Rowling, Stephen H. McLaughlin, Tuomas Knowles and Laura S. Itzhaki

Physical Chemistry Chemical Physics 2014 vol. 16(Issue 14) pp:6448-6459

Publication Date(Web):18 Feb 2014

DOI:10.1039/C3CP54818J

Tandem-repeat proteins, such as leucine-rich repeats, comprise arrays of small structural motifs that pack in a linear fashion to produce elongated architectures. They lack contacts between residues that are distant in primary sequence, a feature that distinguishes them from the complex topologies of globular proteins. Here we have investigated the unfolding pathway of the leucine-rich repeat domain of the mRNA export protein TAP (TAPLRR) using Φ-value analysis. Whereas most of the tandem-repeat proteins studied to date have been found to unfold via a polarised mechanism in which only a small, localised number of repeats are structured in the transition state, the unfolding mechanism of TAPLRR is more diffuse in nature. In the transition state for unfolding of TAPLRR, three of the four LRRs are highly structured and non-native interactions are formed within the N-terminal α-helical cap and the first LRR. Thus, the α-helical cap plays an important role in which non-native interactions are required to provide a scaffold for the LRRs to pack against in the folding reaction.

Polymer physics inspired approaches for the study of the mechanical properties of amyloid fibrils

Co-reporter:Lisa R. Volpatti ;Tuomas P. J. Knowles

Journal of Polymer Science Part B: Polymer Physics 2014 Volume 52( Issue 4) pp:281-292

Publication Date(Web):

DOI:10.1002/polb.23428

ABSTRACT

Amyloid structures constitute a class of highly ordered nanomaterials formed by insoluble protein aggregates. These aggregates are characterized by a cross-β structural motif in which β-sheets are oriented perpendicular to the fibril axis and bound together by a dense hydrogen bonding network. Although they have been associated with several neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, amyloid fibrils have also been found in many physiologically beneficial roles, for instance in adhesives and hormone storage. Inspired by this natural occurrence of functional amyloid, the hierarchal self-assembly of these structures has recently been used to develop artificial biomaterials for applications in medicine and nanotechnology. In order to realize the full potential of amyloids as functional materials, it is important to understand their fundamental mechanical properties. This review explores a range of experimental strategies to determine the mechanical properties of amyloid fibrils and discusses the results in the context of polymer physics concepts. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 281–292

Quantitative Analysis of Diffusive Reactions at the Solid–Liquid Interface in Finite Systems

Co-reporter:Thomas C. T. Michaels, Alexander K. Buell, Eugene M. Terentjev, and Tuomas P. J. Knowles

The Journal of Physical Chemistry Letters 2014 Volume 5(Issue 4) pp:695-699

Publication Date(Web):January 24, 2014

DOI:10.1021/jz4024833

A central element in many processes in physics, chemistry and biology is a reaction between a species immobilized on a surface and a partner that is able to diffuse in solution. However, integrated rate laws for this class of chemical processes have so far only been found in certain special cases. Here, we present a model for the time dependence of an irreversible reaction between particles in a solution of finite volume and a surface. The resulting analytical expression allows quantitative analysis of the transient kinetics of the reaction between soluble particles and a surface. We apply this approach to the analysis of quartz crystal microbalance experiments of protein aggregation under conditions where both reaction and diffusion define the overall kinetics. Furthermore, we use the model to determine absolute mass sensitivity coefficients for soft and rough surfaces, a situation where conventional approaches to determine the mass sensitivity a priori fail.Keywords: amyloid fibers; Cottrell equation; depletion; mass sensitivity; quartz crystal microbalance (QCM); Sauerbrey equation; surface;

Expanding the Solvent Chemical Space for Self-Assembly of Dipeptide Nanostructures

Co-reporter:Thomas O. Mason, Dimitri Y. Chirgadze, Aviad Levin, Lihi Adler-Abramovich, Ehud Gazit, Tuomas P. J. Knowles, and Alexander K. Buell

ACS Nano 2014 Volume 8(Issue 2) pp:1243

Publication Date(Web):January 14, 2014

DOI:10.1021/nn404237f

Nanostructures composed of short, noncyclic peptides represent a growing field of research in nanotechnology due to their ease of production, often remarkable material properties, and biocompatibility. Such structures have so far been almost exclusively obtained through self-assembly from aqueous solution, and their morphologies are determined by the interactions between building blocks as well as interactions between building blocks and water. Using the diphenylalanine system, we demonstrate here that, in order to achieve structural and morphological control, a change in the solvent environment represents a simple and convenient alternative strategy to the chemical modification of the building blocks. Diphenylalanine (FF) is a dipeptide capable of self-assembly in aqueous solution into needle-like hollow micro- and nanocrystals with continuous nanoscale channels that possess advantageous properties such as high stiffness and piezoelectricity and have so emerged as attractive candidates for functional nanomaterials. We investigate systematically the solubility of diphenylalanine in a range of organic solvents and probe the role of the solvent in the kinetics of self-assembly and the structures of the final materials. Finally, we report the crystal structure of the FF peptide in microcrystalline form grown from MeOH solution at 1 Å resolution and discuss the structural changes relative to the conventional materials self-assembled in aqueous solution. These findings provide a significant expansion of the structures and morphologies that are accessible through FF self-assembly for existing and future nanotechnological applications of this peptide. Solvent mediation of molecular recognition and self-association processes represents an important route to the design of new supramolecular architectures deriving their functionality from the nanoscale ordering of their components.Keywords: biomaterials; dipeptides; diphenylalanine; nanotubes; self-assembly; solvatomorphism

Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay

Co-reporter:Paolo Arosio ; Risto Cukalevski ; Birgitta Frohm ; Tuomas P. J. Knowles ;Sara Linse

Journal of the American Chemical Society 2013 Volume 136(Issue 1) pp:219-225

Publication Date(Web):December 6, 2013

DOI:10.1021/ja408765u

The aggregation of the amyloid beta peptide, Aβ42, implicated in Alzheimer’s disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar Aβ42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.

Single-Molecule Measurements of Transient Biomolecular Complexes through Microfluidic Dilution

Co-reporter:Mathew H. Horrocks, Luke Rajah, Peter Jönsson, Magnus Kjaergaard, Michele Vendruscolo, Tuomas P. J. Knowles, and David Klenerman

Analytical Chemistry 2013 Volume 85(Issue 14) pp:6855

Publication Date(Web):June 19, 2013

DOI:10.1021/ac4010875

Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the challenge of observing weakly bound complexes in single-molecule experiments in solution, we have designed a microfluidic device that rapidly dilutes samples by up to one hundred thousand times, allowing the observation of unstable complexes before they dissociate. The device can interface with standard biochemistry laboratory experiments and generates a spatially uniform dilution that is stable over time allowing the quantification of the relative concentrations of different molecular species.

A Clear View of Polymorphism, Twist, and Chirality in Amyloid Fibril Formation

Co-reporter:Lisa R. Volpatti, Michele Vendruscolo, Christopher M. Dobson, and Tuomas P. J. Knowles

ACS Nano 2013 Volume 7(Issue 12) pp:10443

Publication Date(Web):December 23, 2013

DOI:10.1021/nn406121w

The self-assembly of protein molecules into highly ordered linear aggregates, known as amyloid fibrils, is a phenomenon receiving increasing attention because of its biological roles in health and disease and the potential of these structures to form artificial proteinaceous scaffolds for biomaterials applications. A particularly powerful approach to probe the key physical properties of fibrillar structures is atomic force microscopy, which was used by Usov et al. in this issue of ACS Nano to reveal the polymorphic transitions and chirality inversions of amyloid fibrils in unprecedented detail. Starting from this study, this Perspective highlights recent progress in understanding the dynamic polymorphism, twisting behavior, and handedness of amyloid fibrils and discusses the promising future of these self-assembling structures as advanced functional materials with applications in nanotechnology and related fields.

Probing small molecule binding to amyloid fibrils

Co-reporter:Alexander K. Buell, Elin K. Esbjörner, Patrick J. Riss, Duncan A. White, Franklin I. Aigbirhio, Gergely Toth, Mark E. Welland, Christopher M. Dobson and Tuomas P. J. Knowles

Physical Chemistry Chemical Physics 2011 vol. 13(Issue 45) pp:20044-20052

Publication Date(Web):17 Oct 2011

DOI:10.1039/C1CP22283J

Much effort has focussed in recent years on probing the interactions of small molecules with amyloid fibrils and other protein aggregates. Understanding and control of such interactions are important for the development of diagnostic and therapeutic strategies in situations where protein aggregation is associated with disease. In this perspective article we give an overview over the toolbox of biophysical methods for the study of such amyloid-small molecule interactions. We discuss in detail two recently developed techniques within this framework: linear dichroism, a promising extension of the more traditional spectroscopic techniques, and biosensing methods, where surface-bound amyloid fibrils are exposed to solutions of small molecules. Both techniques rely on the measurement of physical properties that are very directly linked to the binding of small molecules to amyloid aggregates and therefore provide an attractive route to probe these important interactions.

Position-Dependent Electrostatic Protection against Protein Aggregation

Co-reporter:Alexer K. Buell;Gian Gaetano Tartaglia Dr.;Neil R. Birkett Dr.;Christopher A. Waudby;Michele Vendruscolo Dr.;Xavier Salvatella Dr.;Mark E. Well ;Tuomas P. J. Knowles Dr.

ChemBioChem 2009 Volume 10( Issue 8) pp:1309-1312

Publication Date(Web):

DOI:10.1002/cbic.200900144

Chemical kinetics for drug discovery to combat protein aggregation diseases

Co-reporter:Paolo Arosio, Michele Vendruscolo, Christopher M. Dobson, Tuomas P.J. Knowles

Trends in Pharmacological Sciences (March 2014) Volume 35(Issue 3) pp:127-135

Publication Date(Web):1 March 2014

DOI:10.1016/j.tips.2013.12.005

•Protein misfolding diseases largely lack effective pharmaceutical treatments.•Compounds with the ability to interfere with protein aggregation are attractive drug candidates.•The search for inhibitors requires a detailed understanding of the inhibition mechanisms.•Chemical kinetic analysis plays a key role in identifying the inhibition mechanisms.Protein misfolding diseases are becoming increasingly prevalent, yet there are very few effective pharmacological treatments. The onset and progression of these diseases is associated with the aberrant aggregation of normally soluble proteins and peptides into amyloid fibrils. Because genetic and physiological findings suggest that protein aggregation is a key event in pathogenesis, an attractive therapeutic strategy against this class of disorders is the search for compounds able to interfere with this process, in particular by suppressing the formation of soluble toxic oligomeric aggregates. In this review, we discuss how chemical kinetics can contribute to the fundamental understanding of the molecular mechanism of aggregation, and speculate on the implications for the development of therapeutic molecules that inhibit specific steps in the aggregation pathway that are crucial for preventing toxicity.

Electrostatic Effects in Filamentous Protein Aggregation

Co-reporter:Alexander K. Buell, Peter Hung, Xavier Salvatella, Mark E. Welland, Christopher M. Dobson, Tuomas P.J. Knowles

Biophysical Journal (5 March 2013) Volume 104(Issue 5) pp:

Publication Date(Web):5 March 2013

DOI:10.1016/j.bpj.2013.01.031

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules.

Biosensor-based label-free assays of amyloid growth

Co-reporter:Duncan A. White, Alexander K. Buell, Christopher M. Dobson, Mark E. Welland, Tuomas P.J. Knowles

FEBS Letters (20 August 2009) Volume 583(Issue 16) pp:2587-2592

Publication Date(Web):20 August 2009

DOI:10.1016/j.febslet.2009.06.008

Uncontrolled fibrous protein aggregation is implicated in a range of aberrant biological phenomena. Much effort has consequently been directed towards establishing quantitative in vitro assays of this process with the aim of probing amyloid growth in molecular detail as well as elucidating the effect of additional species on this reaction. In this paper, we discuss some recent approaches based on label-free technologies focussed on achieving these objectives. Several biosensor techniques have been developed to monitor biomolecular assembly without the requirement for fluorophore marker molecules; in particular quartz crystal microbalance and surface plasmon resonance measurements provide advantageous alternatives to traditional spectroscopic methods and are currently receiving increasing attention in the context of amyloid growth assays.

Role of Elongation and Secondary Pathways in S6 Amyloid Fibril Growth

Co-reporter:Nikolai Lorenzen, Samuel I.A. Cohen, Søren B. Nielsen, Therese W. Herling, Gunna Christiansen, Christopher M. Dobson, Tuomas P.J. Knowles, Daniel Otzen

Biophysical Journal (2 May 2012) Volume 102(Issue 9) pp:

Publication Date(Web):2 May 2012

DOI:10.1016/j.bpj.2012.03.047

The concerted action of a large number of individual molecular level events in the formation and growth of fibrillar protein structures creates a significant challenge for differentiating between the relative contributions of different self-assembly steps to the overall kinetics of this process. The characterization of the individual steps is, however, an important requirement for achieving a quantitative understanding of this general phenomenon which underlies many crucial functional and pathological pathways in living systems. In this study, we have applied a kinetic modeling approach to interpret experimental data obtained for the aggregation of a selection of site-directed mutants of the protein S6 from Thermus thermophilus. By studying a range of concentrations of both the seed structures, used to initiate the reaction, and of the soluble monomer, which is consumed during the growth reaction, we are able to separate unambiguously secondary pathways from primary nucleation and fibril elongation. In particular, our results show that the characteristic autocatalytic nature of the growth process originates from secondary processes rather than primary nucleation events, and enables us to derive a scaling law which relates the initial seed concentration to the onset of the growth phase.

Automated Ex Situ Assays of Amyloid Formation on a Microfluidic Platform

Co-reporter:Kadi-Liis Saar, Emma V. Yates, Thomas Müller, Séverine Saunier, Christopher M. Dobson, Tuomas P.J. Knowles

Biophysical Journal (2 February 2016) Volume 110(Issue 3) pp:

Publication Date(Web):2 February 2016

DOI:10.1016/j.bpj.2015.11.3523

Increasingly prevalent neurodegenerative diseases are associated with the formation of nanoscale amyloid aggregates from normally soluble peptides and proteins. A widely used strategy for following the aggregation process and defining its kinetics involves the use of extrinsic dyes that undergo a spectral shift when bound to β-sheet-rich aggregates. An attractive route to carry out such studies is to perform ex situ assays, where the dye molecules are not present in the reaction mixture, but instead are only introduced into aliquots taken from the reaction at regular time intervals to avoid the possibility that the dye molecules interfere with the aggregation process. However, such ex situ measurements are time-consuming to perform, require large sample volumes, and do not provide for real-time observation of aggregation phenomena. To overcome these limitations, here we have designed and fabricated microfluidic devices that offer continuous and automated real-time ex situ tracking of the protein aggregation process. This device allows us to improve the time resolution of ex situ aggregation assays relative to conventional assays by more than one order of magnitude. The availability of an automated system for tracking the progress of protein aggregation reactions without the presence of marker molecules in the reaction mixtures opens up the possibility of routine noninvasive study of protein aggregation phenomena.

A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions

Co-reporter:Therese W. Herling, David J. O’Connell, Mikael C. Bauer, Jonas Persson, Ulrich Weininger, Tuomas P.J. Knowles, Sara Linse

Biophysical Journal (10 May 2016) Volume 110(Issue 9) pp:

Publication Date(Web):10 May 2016

DOI:10.1016/j.bpj.2016.03.038

The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.

From Macroscopic Measurements to Microscopic Mechanisms of Protein Aggregation

Co-reporter:Samuel I.A. Cohen, Michele Vendruscolo, Christopher M. Dobson, Tuomas P.J. Knowles

Journal of Molecular Biology (10 August 2012) Volume 421(Issues 2–3) pp:160-171

Publication Date(Web):10 August 2012

DOI:10.1016/j.jmb.2012.02.031

The ability to relate bulk experimental measurements of amyloid formation to the microscopic assembly processes that underlie protein aggregation is critical in order to achieve a quantitative understanding of this complex phenomenon. In this review, we focus on the insights from classical and modern theories of linear growth phenomena and discuss how theory allows the roles of growth and nucleation processes to be defined through the analysis of experimental in vitro time courses of amyloid formation. Moreover, we discuss the specific signatures in the time course of the reactions that correspond to the actions of primary and secondary nucleation processes, and outline strategies for identifying and characterising the nature of the dominant process responsible in each case for the generation of new aggregates. We highlight the power of a global analysis of experimental time courses acquired under different conditions, and discuss how such an analysis allows a rigorous connection to be established between the macroscopic measurements and the rates of the individual microscopic processes that underlie the phenomenon of amyloid formation.Download high-res image (172KB)Download full-size imageHighlights► We review classical and modern theories of filamentous growth. ► We outline the application of kinetic theory to the analysis of protein aggregation. ► We highlight the power of rate laws in relating observables to mechanisms. ► We explain connections between macroscopic measurements and microscopic mechanisms. ► We discuss practical requirements for revealing the mechanisms of amyloid formation.

Modulation of electrostatic interactions to reveal a reaction network unifying the aggregation behaviour of the Aβ42 peptide and its variants

Co-reporter:Georg Meisl, Xiaoting Yang, Christopher M. Dobson, Sara Linse and Tuomas P. J. Knowles

Chemical Science (2010-Present) 2017 - vol. 8(Issue 6) pp:NaN4362-4362

Publication Date(Web):2017/04/26

DOI:10.1039/C7SC00215G

The aggregation of the amyloid β peptide (Aβ42), which is linked to Alzheimer's disease, can be altered significantly by modulations of the peptide's intermolecular electrostatic interactions. Variations in sequence and solution conditions have been found to lead to highly variable aggregation behaviour. Here we modulate systematically the electrostatic interactions governing the aggregation kinetics by varying the ionic strength of the solution. We find that changes in the solution ionic strength induce a switch in the reaction pathway, altering the dominant mechanisms of aggregate multiplication. This strategy thereby allows us to continuously sample a large space of different reaction mechanisms and develop a minimal reaction network that unifies the experimental kinetics under a wide range of different conditions. More generally, this universal reaction network connects previously separate systems, such as charge mutants of the Aβ42 peptide, on a continuous mechanistic landscape, providing a unified picture of the aggregation mechanism of Aβ42.

Micro- and nanoscale hierarchical structure of core–shell protein microgels

Co-reporter:Lisa R. Volpatti, Ulyana Shimanovich, Francesco Simone Ruggeri, Sreenath Bolisetty, Thomas Müller, Thomas O. Mason, Thomas C. T. Michaels, Raffaele Mezzenga, Giovanni Dietler and Tuomas P. J. Knowles

Journal of Materials Chemistry A 2016 - vol. 4(Issue 48) pp:NaN7999-7999

Publication Date(Web):2016/11/14

DOI:10.1039/C6TB02683D

Protein nanofibrils were first discovered in the context of misfolding and neurodegenerative diseases but have recently been found in naturally occurring functional materials including algal adhesives, bacterial coatings, and even mammalian melanosomes. These physiologically beneficial roles have led to the exploration of their use as the basis for artificial protein-based functional materials for a range of applications as bioscaffolds and carrier agents. In this work, we fabricate core–shell protein microgels stabilized by protein fibrillation with hierarchical structuring on scales ranging from a few nanometers to tens of microns. With the aid of droplet microfluidics, we exploit fibrillar protein self-assembly together with the aqueous phase separation of a polysaccharide and polyethylene glycol to control the internal structure of the microgels on the micro- and nanoscales. We further elucidate the local composition, morphology, and structural characteristics of the microgels and demonstrate a potential application of core–shell protein microgels for controlling the storage and sequential release of small drug-like molecules. The controlled self-assembly of protein nanofibrils into hierarchical structures can be used in this manner to generate a class of nanomaterials with a range of potential functions and applications.

A microfluidic platform for quantitative measurements of effective protein charges and single ion binding in solution

Co-reporter:Therese W. Herling, Paolo Arosio, Thomas Müller, Sara Linse and Tuomas P. J. Knowles

Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 18) pp:NaN12167-12167

Publication Date(Web):2015/04/16

DOI:10.1039/C5CP00746A

The charge state of proteins in solution is a key biophysical parameter that modulates both long and short range macromolecular interactions. However, unlike in the case of many small molecules, the effective charges of complex biomolecules in solution cannot in general be predicted reliably from their chemical structures alone. Here we present an approach for quantifying the effective charges of solvated biomolecules from independent measurements of their electrophoretic mobilities and diffusion coefficients in free solution within a microfluidic device. We illustrate the potential of this approach by determining the effective charges of a charge-ladder family of mutants of the calcium binding protein calbindin D9k in solution under native conditions. Furthermore, we explore ion-binding under native conditions, and demonstrate the ability to detect the chelation of a single calcium ion through the change that ion binding imparts on the effective charge of calbindin D9k. Our findings highlight the difference between the dry sequence charge and the effective charge of proteins in solution, and open up a route towards rapid and quantitative charge measurements in small volumes in the condensed phase.

Probing small molecule binding to amyloid fibrils

Co-reporter:Alexander K. Buell, Elin K. Esbjörner, Patrick J. Riss, Duncan A. White, Franklin I. Aigbirhio, Gergely Toth, Mark E. Welland, Christopher M. Dobson and Tuomas P. J. Knowles

Physical Chemistry Chemical Physics 2011 - vol. 13(Issue 45) pp:NaN20052-20052

Publication Date(Web):2011/10/17

DOI:10.1039/C1CP22283J

Much effort has focussed in recent years on probing the interactions of small molecules with amyloid fibrils and other protein aggregates. Understanding and control of such interactions are important for the development of diagnostic and therapeutic strategies in situations where protein aggregation is associated with disease. In this perspective article we give an overview over the toolbox of biophysical methods for the study of such amyloid-small molecule interactions. We discuss in detail two recently developed techniques within this framework: linear dichroism, a promising extension of the more traditional spectroscopic techniques, and biosensing methods, where surface-bound amyloid fibrils are exposed to solutions of small molecules. Both techniques rely on the measurement of physical properties that are very directly linked to the binding of small molecules to amyloid aggregates and therefore provide an attractive route to probe these important interactions.

Biophysical approaches for the study of interactions between molecular chaperones and protein aggregates

Co-reporter:Maya A. Wright, Francesco A. Aprile, Paolo Arosio, Michele Vendruscolo, Christopher M. Dobson and Tuomas P. J. Knowles

Chemical Communications 2015 - vol. 51(Issue 77) pp:NaN14434-14434

Publication Date(Web):2015/08/10

DOI:10.1039/C5CC03689E

Molecular chaperones are key components of the arsenal of cellular defence mechanisms active against protein aggregation. In addition to their established role in assisting protein folding, increasing evidence indicates that molecular chaperones are able to protect against a range of potentially damaging aspects of protein behaviour, including misfolding and aggregation events that can result in the generation of aberrant protein assemblies whose formation is implicated in the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The interactions between molecular chaperones and different amyloidogenic protein species are difficult to study owing to the inherent heterogeneity of the aggregation process as well as the dynamic nature of molecular chaperones under physiological conditions. As a consequence, understanding the detailed microscopic mechanisms underlying the nature and means of inhibition of aggregate formation remains challenging yet is a key objective for protein biophysics. In this review, we discuss recent results from biophysical studies on the interactions between molecular chaperones and protein aggregates. In particular, we focus on the insights gained from current experimental techniques into the dynamics of the oligomerisation process of molecular chaperones, and highlight the opportunities that future biophysical approaches have in advancing our understanding of the great variety of biological functions of this important class of proteins.

Diffuse transition state structure for the unfolding of a leucine-rich repeat protein

Co-reporter:Sadie E. Kelly, Georg Meisl, Pamela J. E. Rowling, Stephen H. McLaughlin, Tuomas Knowles and Laura S. Itzhaki

Physical Chemistry Chemical Physics 2014 - vol. 16(Issue 14) pp:

Publication Date(Web):

DOI:10.1039/C3CP54818J