Year 3 research project student-led inquiry labs with productive failure

In year 3 of the UCL Biochemistry BSc / MSci degree, students carry out a 30-credit research project using contemporary techniques. One of the options is to investigate aspects of Protein Structure and Function. This year, we introduced demonstration and inquiry labs. During the initial demonstration labs, students are taught lab techniques and data analysis, learn how these skills can be applied to answer various questions about proteins, and become acquainted with the equipment and proteins available. Rather than being provided with pre-written protocols, they are given selected papers and manufacturer’s product manuals and design their own set of instructions through groupwork with guidance. In Term 2, students use the laboratory for their individual inquiry experiments: they reproduce, modify, or build on existing, self-chosen, peer-reviewed research. They submit their inquiry labs proposal at the end of Term 1, which is either accepted or accepted with alterations based on project quality, instrument availability, safety, and costs. If deemed unsuitable, they can submit another project. During their experiments, they gain hands-on insights into real-life aspects of design, testing, troubleshooting, and reproducibility. They share their experiences, setbacks, and successes in class so others can help and learn. Here, I will describe how student-led experiments, encountering failure, facilitating teacher-student and peer relationships, and focussing on a growth mindset aim to build a sense of ownership and belonging and encourage creativity and exploration

Interplay between HIV Entry and Transportin-SR2 Dependency

<p>Abstract</p> <p>Background</p> <p>Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown.</p> <p>Results</p> <p>Our present analysis of viral specificity reveals that TRN-SR2 is not used to the same extent by all lentiviruses. The DNA flap does not determine the TRN-SR2 requirement of HIV-1. We corroborate the TRN-SR2 independent phenotype of the chimeric HIV virus carrying the MLV capsid and matrix proteins. We reanalyzed the HIV-1 N74D capsid mutant in cells transiently or stably depleted of transportin-SR2 and confirm that the N74D capsid mutant is independent of TRN-SR2 when pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Remarkably, although somewhat less dependent on TRN-SR2 than wild type virus, the N74D capsid mutant carrying the wild type HIV-1 envelope required TRN-SR2 for efficient replication. By pseudotyping with envelopes that mediate pH-independent viral uptake including HIV-1, measles virus and amphotropic MLV envelopes, we demonstrate that HIV-1 N74D capsid mutant viruses retain partial dependency on TRN-SR2. However, this dependency on TRN-SR2 is lost when the HIV N74D capsid mutant is pseudotyped with envelopes mediating pH-dependent endocytosis, such as the VSV-G and Ebola virus envelopes.</p> <p>Conclusion</p> <p>Here we discover a link between the viral entry of HIV and its interaction with TRN-SR2. Our data confirm the importance of TRN-SR2 in HIV-1 replication and argue for careful interpretation of experiments performed with VSV-G pseudotyped viruses in studies on early steps of HIV replication including the role of capsid therein.</p

Structural and Biochemical Investigation of Leucine-Rich Repeat Kinase 2

Parkinson s disease (PD), like other neurodegenerative diseases, is featured by dysfunction and death of cells in selected areas of the nervous system. These hallmarks are guided by various processes caused by genetic, environmental and endogenous factors. Disentangling these strongly interrelated processes to find the primary cause for PD and to separate it from compensatory mechanisms is a demanding scientific task but of great consequence in deciding about possible therapeutic strategies for PD. A real necessity since there is currently no cure for PD.The last years, advances in understanding genetic factors that contribute to PD have provided insights into several possible PD pathogenic mechanisms. One of these genetic factors is the leucine-rich repeat kinase 2 (LRRK2) gene. Mutations in this gene are the most common cause of familial PD and have also been found in apparent sporadic cases of PD, making LRRK2 a popular research subject in the PD science arena.In this thesis, we focused on LRRK2 s structure-function relationship to understand its physiological function and its role in PD. To this end, a combination of in vitro (recombinant protein expression, purification and characterization) and in silico (molecular modelling and bioinformatics) techniques was applied to various research questions namely, LRRK2 s self-regulation mechanisms, the influence of pathogenic mutations on its normal function and specific LRRK2 kinase inhibitors as possible PD therapeutics.Since LRRK2 consists of multiple domains and repeats, its domain folding patterns and their boundaries were determined, a prerequisite for further structure-function analyses, either experimentally or computationally.Subsequently, LRRK2 protein domains were expressed and purified from Escherichia coli (E. coli). The recombinant LRRK2 protein domain that we had most success with was the leucine-rich repeat (LRR) domain. Further characterization of this LRRK2 LRR domain suggests a predominant role in interactions with other LRRK2 domains and / or LRRK2 interacting proteins. A hypothesis that is consistent with our constructed LRRK2 LRR domain homology model showing hydrophobic surface patches that are known to be important in protein-protein interactions. In addition, the putative inter- and / or intramolecular interactions are likely affected by the LRRK2 LRR clinical mutations since we observed little influence of clinical mutant forms on secondary, tertiary or quaternary structure of the LRRK2 LRR domain.Another part of this thesis focused on the LRRK2 kinase active site because several elements indicate that inhibition of LRRK2 kinase activity may constitute a potential therapy for PD. In the absence of a LRRK2 kinase domain experimental structure and in order to aid the development of small-molecule inhibitors against LRRK2 kinase activity, we optimized a LRRK2 kinase domain homology model for use in in silico analysis of LRRK2 kinase inhibitors. The model and docking procedure are valuable for at least three different adenosine triphosphate (ATP)-competitive kinase inhibitor classes with variations both in structure and activity. In addition, preliminary structure-activity relationships identified in this study (i.e. different types of molecular interactions contributing to ATP-competitive kinase inhibitor activity) set a good starting point for the rational design of selective LRRK2 kinase inhibitors.EEN WOORD VAN DANK LIST OF ABBREVIATIONS AND SYMBOLS LIST OF FIGURES LIST OF TABLES SUMMARY SAMENVATTING CHAPTER 1 INTRODUCTION 1.1 PARKINSON’S DISEASE 1.1.1 Clinical features, diagnosis and therapeutic strategies 1.1.2 Etiology 1.1.2.1 Genetic factors 1.1.2.1.1 α-Synuclein 1.1.2.1.2 Parkin 1.1.2.1.3 PINK1 1.1.2.1.4 DJ-1 1.1.2.1.5 LRRK2 1.1.2.2 Non-genetic risk factors 1.1.2.3 Pathogenic mechanisms 1.2 THE ROCO PROTEIN FAMILY 1.2.1 Characterization, diversity and evolution 1.2.2 From biological functions to biochemical mechanisms 1.2.2.1 Biological functions 1.2.2.2 Biochemical mechanisms 1.2.2.2.1 Interacting proteins 1.2.2.2.2 Self-regulation 1.2.2.2.3 External regulation CHAPTER 2 AIMS OF THE STUDY CHAPTER 3 IN SILICO ANALYSIS OF LRRK2 DOMAIN ARCHITECTURE 3.1 MATERIALS AND METHODS 3.2 RESULTS AND DISCUSSION 3.2.1 The N-terminal region 3.2.2 The LRR domain 3.2.3 The ROC-COR bi-domain 3.2.4 The kinase domain 3.2.5 The C-terminal region CHAPTER 4 EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE LRRK2 LRR DOMAIN 4.1 MATERIALS AND METHODS 4.1.1 Materials 4.1.2 Secondary structure and solvent accessibility prediction 4.1.3 Homology modelling 4.1.4 Generation of plasmids 4.1.5 Expression of proteins 4.1.6 Protein fractionation 4.1.7 Purification from soluble protein fraction 4.1.8 Purification from insoluble protein fraction via on column refolding 4.1.9 Size exclusion chromatography – buffer screen 4.1.10 Ion exchange chromatography 4.1.11 Column storage 4.1.12 Column stripping, recharging and / or cleaning 4.1.13 Circular dichroism spectroscopy 4.1.14 Fluorescence spectroscopy 4.1.15 Protein cross-linking 4.1.16 Crystallization screens 4.1.17 Dynamic light scattering 4.1.18 Polyclonal antibody production 4.1.19 SDS–PAGE and Western hybridization 4.2 RESULTS 4.2.1 LRRK2 LRR domain homology model 4.2.2 LRRK2 LRR domain protein expression 4.2.3 LRRK2 LRR domain protein purification 4.2.4 Assessment of LRRK2 LRR domain quaternary structure 4.2.5 Secondary and tertiary structure of the LRRK2 LRR domain 4.2.6 Influence of the LRRK2 LRR domain natural variations 4.2.7 Refolded LRRK2 LRR domain protein was used for the production of polyclonal antibodies 4.2.8 Crystallization screens 4.3 DISCUSSION CHAPTER 5 AN IN VITRO AND IN SILICO STUDY ON SMALL-MOLECULE INHIBITORS OF LRRK2 KINASE ACTIVITY 5.1 MATERIALS AND METHODS 5.1.1 Secondary structure and solvent accessibility prediction 5.1.2 Homology modelling 5.1.2.1 Modelling KINLRRK2 based on multiple templates 5.1.2.2 Modelling ligands in the KINLRRK2 binding site 5.1.3 Database preparation 5.1.4 Protein-ligand docking and scoring 5.1.5 ROC-curves 5.1.6 In vitro kinase reactions 5.2 RESULTS 5.2.1 LRRK2 kinase structural model 5.2.2 LRRK2 kinase - ligand interactions 5.2.2.1 In vitro data 5.2.2.2 In silico data 5.3 DISCUSSION CHAPTER 6 GENERAL CONCLUSIONS AND PERSPECTIVES BIBLIOGRAPHY APPENDIX (SEE CD-ROM) CURRICULUM VITAEnrpages: 179status: publishe

Development of a range of fluorescent reagentless biosensors for ATP, based on malonyl-coenzyme A synthetase

<div><p>The range of ATP concentrations that can be measured with a fluorescent reagentless biosensor for ATP has been increased by modulating its affinity for this analyte. The ATP biosensor is an adduct of two tetramethylrhodamines with MatB from <i>Rhodopseudomonas palustris</i>. Mutations were introduced into the binding site to modify ATP binding affinity, while aiming to maintain the concomitant fluorescence signal. Using this signal, the effect of mutations in different parts of the binding site was measured. This mutational analysis revealed three variants in particular, each with a single mutation in the phosphate-binding loop, which had potentially beneficial changes in ATP binding properties but preserving a fluorescence change of ~3-fold on ATP binding. Two variants (T167A and T303A) weakened the binding, changing the dissociation constant from the parent’s 6 μM to 123 μM and 42 μM, respectively. Kinetic measurements showed that the effect of these mutations on affinity was by an increase in dissociation rate constants. These variants widen the range of ATP concentration that can be measured readily by this biosensor to >100 μM. In contrast, a third variant, S170A, decreased the dissociation constant of ATP to 3.8 μM and has a fluorescence change of 4.2 on binding ATP. This variant has increased selectivity for ATP over ADP of >200-fold. This had advantages over the parent by increasing sensitivity as well as increasing selectivity during ATP measurements in which ADP is present.</p></div

Fluorescence change and affinity for ATP binding to variants of Rho-MatB.

<p>Fluorescence change and affinity for ATP binding to variants of Rho-MatB.</p

Nucleotide affinity for variants of Rho-MatB.

<p>Titration of ATP (circles) and ADP (triangles) to (A) 0.5 µM Rho-MatB T167A, (B) 0.5 μM Rho-MatB T303A; (C) 0.5 μM Rho-MatB S170A. Measurements are shown for one representative experiment and were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 20°C. Excitation was at 553 nm, emission at 571 nm. The dissociation constants and fluorescence ratios were obtained using a quadratic binding equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#sec002" target="_blank">Methods</a>) and are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#pone.0179547.t002" target="_blank">Table 2</a>. The drop lines are to indicate the dissociation constants. Data are normalized to the intensity in the absence of nucleotide.</p

Fluorescence changes, dissociation constants and rate constants for ATP binding to variants of Rho-MatB.

<p>Fluorescence changes, dissociation constants and rate constants for ATP binding to variants of Rho-MatB.</p

Test assay, measurement of the <i>K</i>_m of ADP with pyruvate kinase using T303A variant ATP biosensor.

<p>The assay was run in multiwell format in 50 mM HEPES buffer pH 7.2, 100 mM KCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 25°C. The assay components were 500 μM phosphoenolpyruvate, 0.005 unit ml^-1 pyruvate kinase, 1 μM T303A rho-MatB and the micromolar concentrations of ADP shown on the inset panel for an illustrative set of traces. The data are shown as ATP production rates, using calibrations done at 0–20 μM ATP at 0 μM ADP and with ADP to make 500 μM total nucleotide concentration. Intermediate calibrations were by interpolation. The average (with standard errors) are plotted for four independent measurements, giving <i>K</i>_m of 56 ± 4 μM.</p

Association kinetics of ATP binding to Rho-MatB variants.

<p>Fluorescence time courses were measured by rapidly mixing 0.25 µM Rho-MatB with different concentrations of ATP in large excess (micromolar concentrations shown). Measurements for one representative dataset are shown and were obtained in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 20°C. Time courses were obtained for two different time scales to show fast and slow phases. Slow phases are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#pone.0179547.s003" target="_blank">S3 Fig</a>. Note that the dead time of the stopped-flow instrument (the time taken for the mixed solution to reach the fluorescence observation chamber) is ~2 ms, so that the traces of the fast phase only show changes from that time. (A) Example traces for Rho-MatB T167A, T303A and S170A variants. (B) The average, observed rate constants are shown with standard errors. The fast phases were fit to single exponentials to give rate constants (<i>k</i>_obs), increasing linearly with ATP concentration. “+AMP” is a set of measurements with the parent Rho-MatB in the presence of 500 µM AMP. (C) Conformational selection model for binding derived for Rho-MatB: step 1 is a conformation change of the apoprotein, this is followed by ATP binding. The star indicates the high fluorescence state. Pseudo-first order conditions used for the kinetic measurements give <i>k</i>_obs = <i>k</i>₊₂[ATP] + <i>k</i>_-2: the slope (second order rate constant for association, <i>k</i>₊₂) and intercept (dissociation rate constant, <i>k</i>_-2) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179547#pone.0179547.t002" target="_blank">Table 2</a>.</p

Fluorescence excitation and emission spectra of variants of Rho-MatB.

<p>(A) 1 µM Rho-MatB T167A with and without 5 mM ATP; (B) 1 μM Rho-MatB T303A with and without 3 mM ATP; (C) 1 μM Rho-MatB S170A with and without 0.5 mM ATP. These ATP concentrations were saturating for the variant. Solutions were in 50 mM Hepes pH 7.0, 100 mM NaCl, 10 mM MgCl₂, 0.3 mg ml^-1 bovine serum albumin at 20°C. Spectra are normalized to the maximum intensity in the absence of ATP.</p