Dr Neil Kad
In my lab we use single molecule techniques to understand the physical basis of how proteins interact. A number of diseases are linked to alterations in these physical parameters and we aim to find solutions to these problems.
- DNA Repair
- Muscle Contraction
- Neurodegenerative Disease
We have been funded by the BBSRC, Parkinson's UK, British Heart Foundation, Royal Society and the STFC.
Link to External lab home page
Neil Kad joined the School of Biosciences in August 2014, his previous positions were:
- 2007-2014 – Lecturer, Department of Biological Sciences, University of Essex
- 2004-2007 – Research Associate (PI: Prof. DM. Warshaw), Department of Molecular Physiology and Biophysics, University of Vermont, Vermont USA.
- 2001-2004 - Postdoctoral Research Associate (PI: Prof. DM. Warshaw), Department of Molecular Physiology and Biophysics, University of Vermont, Vermont USA.
- 1998-2001 – Postdoctoral Research Associate (PI: Prof. SE. Radford), Department of Biochemistry, University of Leeds, Leeds UK.
- 1994-1998 - Ph.D. in the conformational kinetics of the chaperonins GroEL and GroES (PI: Prof. AR. Clarke). Department of Biochemistry, University of Bristol
- 1991-1994 - B.Sc. (Hons) Biochemistry. University of Sheffield
Investigating the function of motile enzymes on their tracks. I have two main research foci, the first involves looking at DNA repair and the second myosin function. The latter is the motor enzyme responsible for a number of cellular tasks from muscle contraction to cargo transport.
My key research areas are:
- DNA repair
- Single Molecule Biophysics
- Muscle Contractility Amyloid disease and inhibition
- Molecular Motors
To study these enzymes we use single molecule techniques, in particular Fluorescence Microscopy and a technique called tightroping, where we suspend nanowires of DNA or actin between surface immobilised pedestals.
We label our proteins with quantum dots, these are small nanocrystals that fluoresce brightly and are extremely resistant to photobleaching, making them ideal tools in the study of biological processes.
Using these approaches we are able to shed light on how the enzymes involved in DNA repair interact with one another and with their DNA substrate.
In addition, we have been able to show how myosin's function is coupled to load, and also how myosin's function is related to its structure.
Finally we are really interested in advanced technologies for probing single molecules, such as the use of nanoprobes in collaboration with the Rutherford Appleton Labs in Harwell.
- BI301 -Enzymes and Introduction to Metabolism: Module convener
- BI629 -Proteins: Structure and Function:
- BI600 -Research project: Module convenor
MSc-R projects available for 2019/20
How are genes activated?
You will investigate how transcription factors assemble on DNA to activate genes and interact with RNA polymerase. We use a unique method where we directly image individual molecules interacting with one another. As part of this masters degree you will learn imaging techniques, molecular biology and biochemistry.
Additional research costs: £1200
Correcting the code of life The basis of cancer is DNA damage, you will investigate how cells protect themselves from damage. This project will use cutting-edge imaging of fluorescently tagged proteins in live bacterial cells. In addition, you will study DNA repair in vitro using techniques unique to our lab. As part of this masters degree you will learn imaging techniques, molecular biology, microbiology and biochemistry.
Additional research costs: £1200
Inchingolo, A. et al. (2019). Revealing the mechanism of how cardiac myosin-binding protein C N-terminal fragments sensitize thin filaments for myosin binding. Proceedings of the National Academy of Sciences [Online]. Available at: https://doi.org/10.1073/pnas.1816480116.Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.
Springall, L. et al. (2018). Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival. Nucleic Acids Research [Online] 46:1256-1265. Available at: https://doi.org/10.1093/nar/gkx1244.Nucleotide excision repair (NER) is the primary mechanism for removal of ultraviolet light (UV)-induced DNA photoproducts and is mechanistically conserved across all kingdoms of life. Bacterial NER involves damage recognition by UvrA2 and UvrB, followed by UvrC-mediated incision either side of the lesion. Here, using a combination of in vitro and in vivo single-molecule studies we show that a UvrBC complex is capable of lesion identification in the absence of UvrA. Single-molecule analysis of eGFP-labelled UvrB and UvrC in living cells showed that UV damage caused these proteins to switch from cytoplasmic diffusion to stable complexes on DNA. Surprisingly, ectopic expression of UvrC in a uvrA deleted strain increased UV survival. These data provide evidence for a previously unrealized mechanism of survival that can occur through direct lesion recognition by a UvrBC complex.
Van Houten, B. and Kad, N. (2018). Single-cell mutagenic responses and cell death revealed in real time. Proceedings of the National Academy of Sciences of the United States of America [Online] 115:7168-7170. Available at: https://doi.org/10.1073/pnas.1808986115.
Barnett, J. and Kad, N. (2018). Understanding the coupling between DNA damage detection and UvrA’s ATPase using bulk and single molecule kinetics. The FASEB Journal [Online]. Available at: https://doi.org/10.1096/fj.201800899R.Nucleotide excision repair (NER) protects cells against diverse types of DNA damage, principally UV
irradiation. In Escherichia coli, damage is recognized by 2 key enzymes: UvrA and UvrB. Despite extensive investigation,
the role of UvrA’s 2 ATPase domains in NER remains elusive. Combining single-molecule fluorescence
microscopy and classic biochemical methods, we have investigated the role of nucleotide binding in UvrA’s kinetic
cycle.Measurement of UvrA’s steady-state ATPase activity shows it is stimulated upon binding DNA (kcat 0.71–1.07/
s). Despite UvrA’s ability to discriminate damage,we find UV-damagedDNA does not alter the steady-state ATPase.
To understand how damage affects UvrA, we studied its binding to DNA under various nucleotide conditions at the
single molecule level. We have found that both UV damage and nucleotide cofactors affect the attached lifetime of
UvrA. In the presence of ATP and UV damage, the lifetime is significantly greater compared with undamaged DNA.
To reconcile these observations, we suggest that UvrA uses negative cooperativity between its ATPase sites that is
gated by damage recognition. Only in the presence of damage is the second site activated, most likely in a sequential
manner.—Barnett, J. T., Kad, N. M. Understanding the coupling between DNA damage detection and UvrA’s
ATPase using bulk and single molecule kinetics.
Kad, N. and Van Houten, B. (2016). DNA repair: Clamping down on copy errors. Nature [Online]. Available at: http://dx.doi.org/10.1038/nature20475.Repair enzymes must communicate across hundreds of nucleotides to undo errors made during DNA replication. Imaging reveals that the enzymes do this by forming a series of ring-like clamps that diffuse along the DNA.
Kong, M. et al. (2016). Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process. Molecular Cell [Online] 64:376-387. Available at: http://doi.org/10.1016/j.molcel.2016.09.005.Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the ?-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of ?-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.
Lin, J. et al. (2016). Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing. Nucleic acids research [Online]. Available at: http://dx.doi.org/10.1093/nar/gkw518.Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.
Simons, M. et al. (2015). Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Scientific reports [Online] 5:18486. Available at: http://doi.org/10.1038/srep18486.In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped "nanoprobe" with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these '"DNA tightropes" can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2's binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level.
Walcott, S. and Kad, N. (2015). Direct Measurements of Local Coupling between Myosin Molecules Are Consistent with a Model of Muscle Activation. PLoS computational biology [Online] 11. Available at: http://www.dx.doi.org/10.1371/journal.pcbi.1004599.Muscle contracts due to ATP-dependent interactions of myosin motors with thin filaments composed of the proteins actin, troponin, and tropomyosin. Contraction is initiated when calcium binds to troponin, which changes conformation and displaces tropomyosin, a filamentous protein that wraps around the actin filament, thereby exposing myosin binding sites on actin. Myosin motors interact with each other indirectly via tropomyosin, since myosin binding to actin locally displaces tropomyosin and thereby facilitates binding of nearby myosin. Defining and modeling this local coupling between myosin motors is an open problem in muscle modeling and, more broadly, a requirement to understanding the connection between muscle contraction at the molecular and macro scale. It is challenging to directly observe this coupling, and such measurements have only recently been made. Analysis of these data suggests that two myosin heads are required to activate the thin filament. This result contrasts with a theoretical model, which reproduces several indirect measurements of coupling between myosin, that assumes a single myosin head can activate the thin filament. To understand this apparent discrepancy, we incorporated the model into stochastic simulations of the experiments, which generated simulated data that were then analyzed identically to the experimental measurements. By varying a single parameter, good agreement between simulation and experiment was established. The conclusion that two myosin molecules are required to activate the thin filament arises from an assumption, made during data analysis, that the intensity of the fluorescent tags attached to myosin varies depending on experimental condition. We provide an alternative explanation that reconciles theory and experiment without assuming that the intensity of the fluorescent tags varies.
Desai, R., Geeves, M. and Kad, N. (2015). Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments. Journal of Biological Chemistry [Online]:jbc.M114.609743-jbc.M114.609743. Available at: http://dx.doi.org/10.1074/jbc.M114.609743.Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin’s access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here we directly image single molecules of myosin binding to and activating thin filaments. Using this approach the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 further myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off on a binary fashion.
Cheruvara, H., Kad, N. and Mason, J. (2015). Intracellular Screening of a Peptide Library to Derive a Potent Peptide Inhibitor of ?-Synuclein Aggregation. Journal of Biological Chemistry [Online] 290:7426-7435. Available at: http://dx.doi.org/10.1074/jbc.M114.620484.Aggregation of ?-synuclein (?-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71-82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits ?-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of ?-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD.
Kad, N. and Van Houten, B. (2014). Single molecule approaches: watching DNA repair one molecule at a time: Preface. DNA repair [Online] 20:1. Available at: http://dx.doi.org/10.1016/j.dnarep.2014.06.007.
Acerra, N. et al. (2014). Intracellular selection of peptide inhibitors that target disulphide-bridged A?42 oligomers. Protein science : a publication of the Protein Society [Online] 23:1262-1274. Available at: http://dx.doi.org/10.1002/pro.2509.The ?-amyloid (A?) peptide aggregates into a number of soluble and insoluble forms, with soluble oligomers thought to be the primary factor implicated in Alzheimer's disease pathology. As a result, a wide range of potential aggregation inhibitors have been developed. However, in addition to problems with solubility and protease susceptibility, many have inadvertently raised the concentration of these soluble neurotoxic species. Sandberg et al. previously reported a ?-hairpin stabilized variant of A?42 that results from an intramolecular disulphide bridge (A21C/A31C; A?42cc ), which generates highly toxic oligomeric species incapable of converting into mature fibrils. Using an intracellular protein-fragment complementation (PCA) approach, we have screened peptide libraries using E. coli that harbor an oxidizing environment to permit cytoplasmic disulphide bond formation. Peptides designed to target either the first or second ?-strand have been demonstrated to bind to A?42cc , lower amyloid cytotoxicity, and confer bacterial cell survival. Peptides have consequently been tested using wild-type A?42 via ThT binding assays, circular dichroism, MTT cytotoxicity assays, fluorescence microscopy, and atomic force microscopy. Results demonstrate that amyloid-PCA selected peptides function by both removing amyloid oligomers as well as inhibiting their formation. These data further support the use of semirational design combined with intracellular PCA methodology to develop A? antagonists as candidates for modification into drugs capable of slowing or even preventing the onset of AD.
Acerra, N. et al. (2014). Retro-inversal of intracellular selected ?-amyloid-interacting peptides: implications for a novel Alzheimer's disease treatment. Biochemistry [Online] 53:2101-2111. Available at: http://dx.doi.org/10.1021/bi5001257.The aggregation of ?-amyloid (A?) into toxic oligomers is a hallmark of Alzheimer's disease pathology. Here we present a novel approach for the development of peptides capable of preventing amyloid aggregation based upon the previous selection of natural all-l peptides that bind A?1-42. Using an intracellular selection system, successful library members were further screened via competition selection to identify the most effective peptides capable of reducing amyloid levels. To circumvent potential issues arising from stability and protease action for these structures, we have replaced all l residues with d residues and inverted the sequence. These retro-inverso (RI) peptide analogues therefore encompass reversed sequences that maintain the overall topological order of the native peptides. Our results demonstrate that efficacy in blocking and reversing amyloid formation is maintained while introducing desirable properties to the peptides. Thioflavin-T assays, circular dichroism, and oblique angle fluorescence microscopy collectively indicate that RI peptides can reduce amyloid load, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrate modest reductions in cell toxicity. These conclusions are reinforced using Drosophila melanogaster studies to monitor pupal hatching rates and fly locomotor activity in the presence of RI peptides delivered via RI-trans-activating transcriptional activator peptide fusions. We demonstrate that the RI-protein fragment complementation assay approach can be used as a generalized method for deriving A?-interacting peptides. This approach has subsequently led to several peptide candidates being further explored as potential treatments for Alzheimer's disease.
Hughes, C. et al. (2014). Single molecule techniques in DNA repair:a primer. DNA repair [Online] 20:2-13. Available at: http://dx.doi.org/10.1016/j.dnarep.2014.02.003.A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit--searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.
Lin, J. et al. (2014). TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres. Nucleic acids research [Online] 42:2493-2504. Available at: http://dx.doi.org/10.1093/nar/gkt1132.Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (? 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ? 2.8-3.6 ?(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.
Van Houten, B. and Kad, N. (2014). Investigation of bacterial nucleotide excision repair using single-molecule techniques. DNA repair [Online] 20:41-48. Available at: http://dx.doi.org/10.1016/j.dnarep.2013.10.012.Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER.
Acerra, N., Kad, N. and Mason, J. (2013). Combining intracellular selection with protein-fragment complementation to derive A? interacting peptides. Protein engineering, design & selection : PEDS [Online] 26:463-470. Available at: http://dx.doi.org/10.1093/protein/gzt021.Aggregation of the ?-amyloid (A?) peptide into toxic oligomers is considered the primary event in the pathogenesis of Alzheimer's disease. Previously generated peptides and mimetics designed to bind to amyloid fibrils have encountered problems in solubility, protease susceptibility and the population of small soluble toxic oligomers. We present a new method that opens the possibility of deriving new amyloid inhibitors. The intracellular protein-fragment complementation assay (PCA) approach uses a semi-rational design approach to generate peptides capable of binding to A?. Peptide libraries are based on A? regions responsible for instigating amyloidosis, with screening and selection occurring entirely inside Escherichia coli. Successfully selected peptides must therefore bind A? and recombine an essential enzyme while permitting bacterial cell survival. No assumptions are made regarding the mechanism of action for selected binders. Biophysical characterisation demonstrates that binding induces a noticeable reduction in amyloid. Therefore, this amyloid-PCA approach may offer a new pathway for the design of effective inhibitors against the formation of amyloid in general.
Hughes, C. et al. (2013). Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes. Nucleic acids research [Online] 41:4901-4912. Available at: http://dx.doi.org/10.1093/nar/gkt177.Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA-D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB-DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC-DNA complex, we investigated three UvrB mutants: Y96A, a ?-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.
Zhang, C. et al. (2012). A branched kinetic scheme describes the mechanochemical coupling of Myosin Va processivity in response to substrate. Biophysical journal [Online] 103:728-737. Available at: http://dx.doi.org/10.1016/j.bpj.2012.07.033.Myosin Va is a double-headed cargo-carrying molecular motor that moves processively along cellular actin filaments. Long processive runs are achieved through mechanical coordination between the two heads of myosin Va, which keeps their ATPase cycles out of phase, preventing both heads detaching from actin simultaneously. The biochemical kinetics underlying processivity are still uncertain. Here we attempt to define the biochemical pathways populated by myosin Va by examining the velocity, processive run-length, and individual steps of a Qdot-labeled myosin Va in various substrate conditions (i.e., changes in ATP, ADP, and P(i)) under zero load in the single-molecule total internal reflection fluorescence microscopy assay. These data were used to globally constrain a branched kinetic scheme that was necessary to fit the dependences of velocity and run-length on substrate conditions. Based on this model, myosin Va can be biased along a given pathway by changes in substrate concentrations. This has uncovered states not normally sampled by the motor, and suggests that every transition involving substrate binding and release may be strain-dependent.
Dunn, A. et al. (2011). Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA. Nucleic acids research [Online] 39:7487-7498. Available at: http://dx.doi.org/10.1093/nar/gkr459.Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix-hairpin-helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained ?-DNA molecules suspended between 5?µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe(111) insertion.
Kad, N. et al. (2010). Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins. Molecular cell [Online] 37:702-713. Available at: http://dx.doi.org/10.1016/j.molcel.2010.02.003.How DNA repair proteins sort through a genome for damage is one of the fundamental unanswered questions in this field. To address this problem, we uniquely labeled bacterial UvrA and UvrB with differently colored quantum dots and visualized how they interacted with DNA individually or together using oblique-angle fluorescence microscopy. UvrA was observed to utilize a three-dimensional search mechanism, binding transiently to the DNA for short periods (7 s). UvrA also was observed jumping from one DNA molecule to another over approximately 1 microm distances. Two UvrBs can bind to a UvrA dimer and collapse the search dimensionality of UvrA from three to one dimension by inducing a substantial number of UvrAB complexes to slide along the DNA. Three types of sliding motion were characterized: random diffusion, paused motion, and directed motion. This UvrB-induced change in mode of searching permits more rapid and efficient scanning of the genome for damage.
Kad, N., Trybus, K. and Warshaw, D. (2008). Load and Pi control flux through the branched kinetic cycle of myosin V. The Journal of biological chemistry [Online] 283:17477-84. Available at: http://dx.doi.org/10.1074/jbc.M800539200.Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mm phosphate (P(i)), both forward and backward steps become less load-dependent. From these data, we infer that P(i) release commits myosin V to undergo a highly load-dependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of P(i). We propose a branched kinetic model to explain these data. Our model includes P(i) release prior to the most load-dependent step in the cycle, implying that P(i) release and load both act as checkpoints that control the flux through two parallel pathways.
Kad, N. et al. (2007). Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin. Biophysical journal [Online] 92:1623-1631. Available at: http://dx.doi.org/10.1529/biophysj.106.097618.The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the approximately 270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by approximately 20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.
Kad, N. et al. (2005). Single-myosin crossbridge interactions with actin filaments regulated by troponin-tropomyosin. Proceedings of the National Academy of Sciences of the United States of America [Online] 102:16990-16995. Available at: http://dx.doi.org/10.1073/pnas.0506326102.Striated muscle contraction is governed by the thin filament regulatory proteins troponin and tropomyosin. Here, we investigate the molecular mechanisms by which troponin-tropomyosin inhibits myosin's interactions with the thin filament in the absence of calcium by using a laser trap. The displacement events for a single-myosin molecule interacting with a reconstituted thin filament were shorter (step size = 5 nm) and prolonged (69 ms) compared with actin alone (11 nm and 26 ms, respectively). However, these changes alone do not account for the degree of inhibition of thin filament movement observed in an ensemble assay. Our investigations of single- and multiple-myosin molecules with regulated thin filaments suggest the primary basis for this inhibition derives from an approximately 100-fold decrease in the probability of myosin attaching to actin. At higher myosin concentrations, short bursts of motility are observed in a laser trap consistent with the strong binding of a single-myosin crossbridge, resulting in cooperative binding of other cycling crossbridges. We confirmed this cooperativity in the in vitro motility assay by observing thin filament translocation in the absence of calcium but at low [ATP], consistent with rigor activation. We have developed a simple mechanistic model that reproduces and provides insight into both the observed single-myosin molecule and ensemble data in the absence of Ca(2+). These data support the hypothesis that thin filament inhibition in the absence of Ca(2+) is largely achieved by modulating the rate of attachment and/or transition from the weakly to strongly bound state.
Kad, N. et al. (2003). A mutant heterodimeric myosin with one inactive head generates maximal displacement. The Journal of cell biology [Online] 162:481-488. Available at: http://dx.doi.org/10.1083/jcb.200304023.Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402-4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of approximately 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.
Jones, S. et al. (2003). Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro. Journal of molecular biology [Online] 325:249-257. Available at: http://dx.doi.org/10.1016/S0022-2836(02)01227-5.Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.
Kad, N. et al. (2003). Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy. Journal of molecular biology [Online] 330:785-797. Available at: http://dx.doi.org/10.1016/S0022-2836(03)00583-7.The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.
Kad, N. et al. (2001). Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro. Journal of molecular biology [Online] 313:559-571. Available at: http://dx.doi.org/10.1006/jmbi.2001.5071.Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.
McParland, V. et al. (2000). Partially unfolded states of beta(2)-microglobulin and amyloid formation in vitro. Biochemistry [Online] 39:8735-8746. Available at: http://dx.doi.org/10.1021/bi000276j.Dialysis-related amyloidosis (DRA) involves the aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils. Using Congo red and thioflavin-T binding, electron microscopy, and X-ray fiber diffraction, we have determined conditions under which recombinant monomeric beta(2)m spontaneously associates to form fibrils in vitro. Fibrillogenesis is critically dependent on the pH and the ionic strength of the solution, with low pH and high ionic strength favoring fibril formation. The morphology of the fibrils formed varies with the growth conditions. At pH 4 in 0.4 M NaCl the fibrils are approximately 10 nm wide, relatively short (50-200 nm), and curvilinear. By contrast, at pH 1.6 the fibrils formed have the same width and morphology as those formed at pH 4 but extend to more than 600 nm in length. The dependence of fibril growth on ionic strength has allowed the conformational properties of monomeric beta(2)m to be determined under conditions where fibril growth is impaired. Circular dichroism studies show that titration of one or more residues with a pK(a) of 4.7 destabilizes native beta(2)m and generates a partially unfolded species. On average, these molecules retain significant secondary structure and have residual, non-native tertiary structure. They also bind the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid (ANS), show line broadening in one-dimensional (1)H NMR spectra, and are weakly protected from hydrogen exchange. Further acidification destabilizes this species, generating a second, more highly denatured state that is less fibrillogenic. These data are consistent with a model for beta(2)m fibrillogenesis in vitro involving the association of partially unfolded molecules into ordered fibrillar assemblies.
Cliff, M. et al. (1999). A kinetic analysis of the nucleotide-induced allosteric transitions of GroEL. Journal of molecular biology [Online] 293:667-684. Available at: http://dx.doi.org/10.1006/jmbi.1999.3138.Single-point mutants of GroEL were constructed with tryptophan replacing a tyrosine residue in order to examine nucleotide-induced structural transitions spectrofluorometrically. The tyrosine residues at positions 203, 360, 476 and 485 were mutated. Of these, the probe at residue 485 gave the clearest fluorescence signals upon nucleotide binding. The probe at 360 reported similar signals. In response to the binding of ATP, the indole fluorescence reports four distinct structural transitions occurring on well-separated timescales, all of which precede hydrolysis of the nucleotide. All four of these rearrangements were analysed, two in detail. The fastest is an order of magnitude more rapid than previously identified rearrangements and is proposed to be a T-to-R transition. The next kinetic phase is a rearrangement to the open state identified by electron cryo-microscopy and this we designate an R to R* transition. Both of these rearrangements can occur when only a single ring of GroEL is loaded with ATP, and the results are consistent with the occupied ring behaving in a concerted, cooperative manner. At higher ATP concentrations both rings can be loaded with the nucleotide and the R to R* transition is accelerated. The resultant GroEL:ATP14 species can then undergo two final rearrangements, RR*-->[RR](+)-->[RR](#). These final slow steps are completely blocked when ADP occupies the second ring, i.e. it does not occur in the GroEL:ATP7:ADP7 or the GroEL:ATP7 species. All equilibrium and kinetic data conform to a minimal model in which the GroEL ring can exist in five distinct states which then give rise to seven types of oligomeric conformer: TT, TR, TR*, RR, RR*, [RR](+) and [RR](#), with concerted transitions between each. The other eight possible conformers are presumably disallowed by constraints imposed by inter-ring contacts. This kinetic behaviour is consistent with the GroEL ring passing through distinct functional states in a binding-encapsulation-folding process, with the T-form having high substrate affinity (binding), the R-form being able to bind GroES but retaining substrate affinity (encapsulation), and the R*-form retaining high GroES affinity but allowing the substrate to dissociate into the enclosed cavity (folding). ADP induces only one detectable rearrangement (designated T to T*) which has no properties in common with those elicited by ATP. However, asymmetric ADP binding prevents ATP occupying both rings and, hence, restricts the system to the T*T, T*R and T*R* complexes.
Kad, N. et al. (1998). Asymmetry, commitment and inhibition in the GroE ATPase cycle impose alternating functions on the two GroEL rings. Journal of molecular biology [Online] 278:267-278. Available at: http://dx.doi.org/10.1006/jmbi.1998.1704.The ATPase cycle of GroE chaperonins has been examined by transient kinetics to dissect partial reactions in complexes where GroEL is asymmetrically loaded with nucleotides. The occupation of one heptameric ring by ADP does not inhibit the loading of the other with ATP nor does it prevent the consequent structural rearrangement to the "open" state. However, ADP binding completely inhibits ATP hydrolysis in the asymmetric complex, i.e. ATP cannot by hydrolysed when ADP is bound to the other ring. This non-competitive inhibition of the ATPase by ADP is consistent with a ring-switching, or "two-stroke", mechanism of the type: ATP:GroEL --> ADP:GroEL --> ADP:GroEL:ATP --> GroEL:ATP --> GroEL:ADP, i.e. with respect to the GroEL rings, ATP turns over in an alternating fashion. When the ATP-stabilized, "open" state is challenged with hexokinase and glucose, to quench the free ATP, the open state relaxes slowly (0.44 s-1) back to the apo (or closed) conformation. This rate, however, is three times faster than the hydrolytic step, showing that bound ATP is not committed to hydrolysis. When GroES is bound to the GroEL:ATP complex and the system is quenched in the same way, approximately half of the bound ATP undergoes hydrolysis on the chaperonin complex showing that the co-protein increases the degree of commitment. Thus, non-competitive inhibition of ATP hydrolysis, combined with the ability of the co-protein to block ligand exchange between rings has the effect of imposing a reciprocating cycle of reactions with ATP hydrolysing, and GroES binding, on each of the GroEL rings in turn. Taken together, these data imply that the dominant, productive steady state reaction in vivo is: GroEL:ATP:GroES --> GroEL:ADP:GroES --> ATP:GroEL:ADP:GroES --> ATP:GroEL:ADP --> GroES:ATP:GroEL:ADP --> GroES:ATP:GroEL for a hemi-cycle, and that significant inhibi tion of hydrolysis may arise through the formation of a dead-end ADP:GroEL:ATP:GroES complex.
Kong, M. et al. (2017). Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time. in: Methods in Enzymology. Elsevier, pp. 213-257. Available at: https://doi.org/10.1016/bs.mie.2017.03.027.Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein–DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair.
Wang, J. et al. (2017). Integrating Optical Tweezers, DNA Tightropes, and Single-Molecule Fluorescence Imaging: Pitfalls and Traps. in: Single-Molecule Enzymology: Nanomechanical Manipulation and Hybrid Methods. Elsevier, pp. 171-192. Available at: https://doi.org/10.1016/bs.mie.2016.08.003.Fluorescence imaging is one of the cornerstone techniques for understanding how single molecules search for their targets on DNA. By tagging individual proteins, it is possible to track their position with high accuracy. However, to understand how proteins search for targets, it is necessary to elongate the DNA to avoid protein localization ambiguities. Such structures known as "DNA tightropes" are tremendously powerful for imaging target location; however, they lack information about how force and load affect protein behavior. The use of optically trapped microstructures offers the means to apply and measure force effects. Here we describe a system that we recently developed to enable individual proteins to be directly manipulated on DNA tightropes. Proteins bound to DNA can be conjugated with Qdot fluorophores for visualization and also directly manipulated by an optically trapped, manufactured microstructure. Together this offers a new approach to understanding the physical environment of molecules, and the combination with DNA tightropes presents opportunities to study complex biological phenomena.
Springall, L., Inchingolo, A. and Kad, N. (2016). DNA-Protein Interactions Studied Directly Using Single Molecule Fluorescence Imaging of Quantum Dot Tagged Proteins Moving on DNA Tightropes. in: Leake, M. C. ed. Chromosome Architecture. Springer, pp. 141-150. Available at: http://www.dx.doi.org/10.1007/978-1-4939-3631-1_11.Many protein interactions with DNA require specific sequences; however, how these sequences are located remains uncertain. DNA normally appears bundled in solution but, to study DNA-protein interactions, the DNA needs to be elongated. Using fluidics single DNA strands can be efficiently and rapidly elongated between beads immobilized on a microscope slide surface. Such "DNA tightropes" offer a valuable method to study protein search mechanisms. Real-time fluorescence imaging of these interactions provides quantitative descriptions of search mechanism at the single molecule level. In our lab, we use this method to study the complex process of nucleotide excision DNA repair to determine mechanisms of damage detection, lesion removal, and DNA excision.
Kad, N. and Van Houten, B. (2012). Dynamics of lesion processing by bacterial nucleotide excision repair proteins. in: Progress in Molecular Biology and Translational Science. Elsevier, pp. 1-24.Single-molecule approaches permit an unrivalled view of how complex systems operate and have recently been used to understand DNA-protein interactions. These tools have enabled advances in a particularly challenging problem, the search for damaged sites on DNA. DNA repair proteins are present at the level of just a few hundred copies in bacterial cells to just a few thousand in human cells, and they scan the entire genome in search of their specific substrates. How do these proteins achieve this herculean task when their targets may differ from undamaged DNA by only a single hydrogen bond? Here we examine, using single-molecule approaches, how the prokaryotic nucleotide excision repair system balances the necessity for speed against specificity. We discuss issues at a theoretical, biological, and technical level and finally pose questions for future research.
Kad, N. and Radford, S. (2001). Partial unfolding as a precursor to amyloidosis: A discussion of the occurrence, role and implications. in: Lund, P. A. ed. Molecular Chaperones in the Cell. Oxford University Press.
Kad, N. (2019). Program to create Mean Squared displacement data from diffusion. [Text program].This is a Visual Basic macro designed to create Mean-Square Displacement profiles of single molecule diffusion in up to two dimensions. The VBA script should be loaded into Microsoft Excel and x-data should be loaded from the fourth row of the data table in the second column. The first column is time and the third column is for y-data. If only one dimension is required just set the third column to ones all the way down.
The output from this program is time versus MSD.