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DEVELOPMENT OF

-LACTAMASE-BASED BIOSENSOR FOR ANTI-TUBERCULOSIS DRUG

SCREENING

ZOE CHAN

PhD

The Hong Kong Polytechnic University

2019

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The Hong Kong Polytechnic University

Department of Applied Biology and Chemical Technology

Development of -Lactamase-based Biosensor for Anti-tuberculosis Drug

Screening

Zoe Chan

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy

August 2018

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Certificate of Originality

I hereby declare that this thesis is my own work and that, to the best of my knowledge and belief, it reproduces no material previously published or written, nor material that has been accepted for the award of any other degree or diploma, except where due acknowledgement has been made in the text.

Zoe Chan 2018

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To my parents, Elise, Cola, and Colin

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Abstract

Tuberculosis has been one of the deadliest disease in history.

Although it became curable since the discovery of antibiotics, drug resistance was soon developed and the needs in new antituberculosis treatment escalated. In the past, our group has developed an e↵ective -lactamase-based fluorescent biosensor for -lactam antibiotics detection. BlaC, a -lactamase responsible for the -lactam antibiotic resistance in Mycobacterium tuberculosis, was engineered to be a sensitive and efficient biosensor for screening potential inhibitors to be used in combination with currently available -lactam antibiotics.

Thr-216 was mutated to Cys by site-directed mutagenesis and was named T216C. Various fluorophores were labelled on the mutated residue and the prospective fluorescent sensor enzyme was screened.

The functionality of the most promising fluorophore-labelled enzyme T216Cf against antibiotics and inhibitors was investigated with fluorescence spectrometry. Immediate surges in fluorescence intensity of T216Cf upon penicillins addition and positive signals induced by carbapenems and inhibitors were observed, but the response to cephalosporins was insignificant. The relationship between enzyme-inhibitor complex formation and fluorescence intensity change was also revealed by the combination of ESI-MS and fluorescence spectrometry. The kinetic parameters were measured and calculated to examine the impact of mutagenesis and labelling. A second mutation to Trp was introduced at Ile-105 and Thr-237 in an attempt to enhance the biosensor sensitivity. T216Cf/I105W showed a major improvement in sensitivity, as much as from +35% to +83% upon inhibitor addition, although further optimisation can be done on the biosensor efficiency. Molecular dynamics simulations were conducted to understand the mechanism behind the fluorescence signals, which is transferable to similar -lactamase-based biosensor. The change in residues in proximity to the fluorescein, solvent exposure, and interaction with quenchers such as tryptophan all contribute to the fluorescence signals. The promising results suggested that the new T216Cf has great potential to be refined as a powerful biosensor for drug screening.

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Acknowledgements

I would like to thank my supervisor, Prof. Thomas Leung, for his guidance and supervision, and the timely encouragement that has always kept me up. Most importantly, his patience and enthusiasm have truly inspired me as a scientist. This PhD and this thesis would be impossible without him.

And also to my co-supervisor, Prof. Edman Tsang, for the scientific training he has prepared me with and for always watching out for me.

There would surely not be an MD part in this thesis without Dr. Y.

W. Chen, for every bit of my MD knowledge came from him, directly or indirectly. And of course I am most grateful for the necessary co↵ee breaks.

To everyone that helped the development of this project, Dr. H. K. Yap for the clone, Dr. P. K. So for the mass spectrometry measurements, and Dr. W. T. Wong, Sharon, and Emily for the skills and techniques. And also to my group for the past three years.

Needless to say, I am thankful for my parents for everything. Lastly, to Elle, Cola, and Colin, for always being here and supportive.

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List of Figures

Figure 1 The mimicry of -lactam antibiotics to d-alanyl-d-alanine

facilitates their binding to the active site of PBPs. . . 4

Figure 2 Reaction mechanism of -lactamase on ampicillin. . . 10

Figure 3 Hydrogen bond network of the active site of acylated BlaC -lactamase. . . 11

Figure 4 Deacylation of -lactamase on ampicillin. . . 11

Figure 5 Structure of BlaC -lactamase. . . 12

Figure 6 Plasmid map of expression vector pRset-K. . . 27

Figure 7 Elution profile of the nickel affinity chromatography for T216C. . . 49

Figure 8 SDS-PAGE analysis of T216C purification. . . 49

Figure 9 Elution profile of the gel filtration chromatography for T216Ciaf. . . 51

Figure 10 SDS-PAGE analysis of T216Ciaf purification. . . 51

Figure 11 Transformed mass spectrum of T216C and T216Cf. . . 53

Figure 12 CW EPR spectrum of T216C labelled with spin probe, 3-(2-iodoacetamido)-PROXYL. . . 55

Figure 13 Mobility map plotted by Czogalla et al. (2007) of di↵erent regions of protein. . . 56

Figure 14 Steady state fluorescence spectra of 1⇥ 10 ⁶M T216Cf against 4⇥ 10 ³M penicillins. . . 58

Figure 15 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf with di↵erent antibiotics. . . 61

Figure 16 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Ciaf with di↵erent antibiotics. . . 62

Figure 17 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf with the presence of various -lactam antibiotics. . . 65

Figure 18 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf with the presence of di↵erent inhibitors. . . 67

Figure 19 Time-resolved fluorescene spectra of T216Cf against di↵erent concentration of clavulanate. . . 68

Figure 20 ESI-MS study of T216Cf against clavulanate. . . 70

Figure 21 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf with di↵erent antibiotics. . . 72

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Figure 22 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf with multiple penicillins injections. . . 73 Figure 23 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf

with di↵erent inhibitors. . . 74 Figure 24 Time-resolved fluorescene spectra of T216Cf against

di↵erent concentration of BLIP. . . 78 Figure 25 Time-resolved fluorescence spectra of 1⇥ 10 ⁶M T216Cf

with 1.5⇥ 10 ⁶M BLIP. . . 79 Figure 26 R and S enantiomers of F5M-cysteine complex. . . 82 Figure 27 Molecular model of T216Cf showing fluorophore and its

neighbouring residues. . . 83 Figure 28 Molecular model of T216Cf showing fluorophore and

mutated residues. . . 83 Figure 29 Elution profile of the nickel affinity chromatography for

T216C. . . 88 Figure 30 SDS-PAGE analysis of T216C purification. . . 88 Figure 31 SDS-PAGE analysis of labelled BlaC mutants. . . 89 Figure 32 Time-resolved fluorescence spectra of 4⇥ 10 ⁷M labelled

mutants with the presence of various -lactam antibiotics. 93 Figure 33 Time-resolved fluorescence spectra of 4⇥ 10 ⁷M labelled

mutants with the presence of di↵erent inhibitors. . . 95 Figure 34 Extended time-resolved fluorescence spectra of 4⇥ 10 ⁷M

labelled mutants with the presence of di↵erent inhibitors. 96 Figure 35 Time-resolved fluorescene spectra of T216Cf/I105W

against di↵erent concentration of clavulanate. . . 98 Figure 36 torsion angle of cysteine. . . 101 Figure 37 RMSD of each possible models before and after inhibitor

added. . . 107 Figure 38 Molecular model of the covalent complex of T216Cf with

clavulanate at the equilibrium position. . . 108 Figure 39 Chemical structures of fluorescein. . . 110 Figure 40 Hydrophilic solvent accessible surface area of fluorescein. . 112 Figure 41 Molecular model of fluorescein probe attached to T216C at

equilibrium position with and without inhibitor bound. . 113 Figure 42 Projection of charged residues surrounding fluorescein with

and without clavulanate as inhibitor. . . 115 Figure 43 Convention for analysis of geometry of quenching. . . 117

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Figure 44 Geometrical analysis for quenching. . . 118

Figure 45 Fluorescene quenching at t=12770ps. . . 119

Figure 46 TEM-1/BLIP and BlaC/BLIP model structures. . . 124

Figure 47 RMSD of T216Cf/BLIP complex. . . 125

Figure 48 The hydrophilic SASA decreases by 15.5% with BLIP binding to T216Cf. . . 126

Figure 49 Fluorescein partially covered by BLIP and the -lactamase (cyan) in the T216Cf/BLIP complex. . . 126

Figure 50 Crystals of T216Cf formed under Crystal Screen #25 (0.1 M Imidazole pH 6.5, 1.0 M Sodium acetate trihydrate) with 1:1 T216Cf to bu↵er (v/v). . . 129

Figure 51 Crystals of T216Cf formed under Crystal Screen #25 (0.1 M Imidazole pH 6.5, 1.0 M Sodium acetate trihydrate) with 2:1 T216Cf to bu↵er (v/v). . . 130

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List of Tables

Table 1 Common -lactam antibiotics . . . 5 Table 2 Ambler classification of -lactamase . . . 8 Table 3 Peak shift and percentage change in fluorescence intensity

of fluorophore-labelled T216C upon the addition of -lactam antibiotics. . . 59 Table 4 Peak shift and percentage change in fluorescence intensity

of T216Cf upon the addition of -lactam antibiotics. . . . 63 Table 5 Peak shift and percentage change in fluorescence intensity

of T216Ciaf upon the addition of -lactam antibiotics. . . 63 Table 6 Kinetic parameters of T216C and T216Cf mutants for

penicillin G. . . 75 Table 7 Inhibition of T216C and T216Cf mutants by tazobactam,

sulbactam, and clavulanic acid. . . 77 Table 8 Primers for site-directed mutagenesis of T216Cf. . . 84 Table 9 PCR method and conditions for site-directed mutagenesis

of T216Cf. . . 85 Table 10 Percentage change in fluorescence intensity of labelled

-lactamase-based biosensors upon the addition of penicillins and carbapenems. . . 93 Table 11 Percentage change in fluorescence intensity of labelled

-lactamase-based biosensors upon the addition of penicillins and carbapenems. . . 97 Table 12 Starting structures of T216Cf for MD simulations. . . 102 Table 13 Hydrophilic solvent accessible surface area of fluorescein. . 112 Table 14 Charged neighbouring residues of fluorescein with their

distances from the centre of xanthene in fluorescein. . . . 114 Table 15 Geometrical analysis for quenching. . . 118

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Abbreviations

6-IAF 6-Iodoacetamidofluorescein

˚A ˚angstr¨om(s), 10 ¹⁰m

BADAN 6-bromoacetyl-2-dimethylaminoaphthalene

BCA bicinchoninic acid

BLIP -lactamase inhibitor protein

BSA bovine serum albumin

CV column volume(s)

CW EPR continuous wave electron paramagnetic resonance DFT density functional theory

DMF dimethylformamide

DMSO dimethyl sulfoxide DNA deoxyribonucleic acid

dNTP deoxynucleotide triphosphate DOPE discrete optimised protein energy dUTP 2'-deoxyuridine 5'-triphosphate

dUTPase deoxyuridine 5'-triphosphate nucleotidohydrolase E. coli Escherichia coli

ESBL extended-spectrum -lactamase

ESI-MS electrospray ionization mass spectrometry F5M fluorescein-5-maleimide

FPLC fast protein liquid chromatography HIV human immunodeficiency virus

h hour(s)

IC₅₀ half maximal inhibitory concentration IPTG isopropythiogalactoside

kDa kilodalton(s)

LB lysogeny broth

M. tuberculosis Mycobacterium tuberculosis mAU milli-absorption unit(s)

min minute(s)

MD molecular dynamics

MDR multidrug-resistant

MM molecular mechanics

NMR nuclear magnetic resonance

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NMWL nominal molecular weight limit

OD optical density

PBP penicillin-binding protein PCR polymerase chain reaction

PDB Protein Data Bank

PME Particle-mesh Ewald

PROXYL 2,2,5,5-tetramethyl-1-pyrrolidinyloxy RMSD root-mean-square deviation

rpm round per minute

RR rifampicin-resistant

s second(s)

SASA solvent accessible surface area

SDS sodium dodecyl sulphate

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDSL site-directed spin labelling

SQM semi-empirical quantum mechanical

TB tuberculosis

TMR5M tetramethylrhodamine-5-maleimide

UV ultraviolet

v/v volume per volume

w/v weight per volume

XDR extensively drug-resistant

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1 Introduction

1.1 Mycobacterium tuberculosis

For thousands of years, human has been battling with tuberculosis (TB).

From as early as the Neolithic, TB has haunted mankind intercontinentally for its notorious high mortality rate (Bos et al., 2014; Daniel, 2006; Donoghue et al., 2010; Hershkovitz et al., 2008; Salo et al., 1994; Wirth et al., 2008).

The case fatality rate of TB was approximated to be as high as 70% in the pre-chemotherapy era (Tiemersma et al., 2011). Despite being preventable with early diagnosis and curable with prompt and appropriate treatment, TB remains one of the top ten causes of mortality. Worldwide, it is estimated that 6.3 million new cases were diagnosed and 1.6 million died from TB in the year 2016 (World Health Organization, 2017). Although Koch first reported his discovery of Mycobacterium tuberculosis and identified the cause of TB in 1882 (Koch, 1882), there was no efficient drug treatment until 1943 when Schatz et al. (1944) isolated streptomycin, the first antibiotic cure for TB (Schatz et al., 1944). After that, research on mycobactericidal drug bloomed and antibiotics have been the most common treatment to TB.

Nowadays, rifampicin and isoniazid are the most e↵ective amongst anti-TB drugs and are part of the first-line treatment (World Health Organization, 2017). However, over time, the bacterium has largely evolved and resistance to antibiotics has been developed. For instance, 4.1% of the new cases and 19% of the previously treated cases had either rifampicin-resistant

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TB or multidrug-resistant TB (MDR/RR-TB, resistant to rifampicin or to both rifampicin and isoniazid) in 2016. Nearly 60 thousand cases of extensively drug-rsistant TB (XDR-TB, multidrug-resistant TB that is also resistant to fluoroquinoline and second-line injectable agent) has also been reported in the year of 2016 by over a hundred countries. Whilst the overall treatment success rate for TB is 91%, the success rates for MDR/RR-TB and for XDR-TB remain low, with 54% and 30% respectively (World Health Organization, 2016). In recent years, TB patients described to have

“totally”drug-resistant TB (strains that showed in vitro resistance to all first- and second-line drugs tested) were identified in South Africa, India, Iran, and Italy (Migliori et al., 2007; Slomski, 2013; Udwadia et al., 2012;

Velayati et al., 2009). Indubitably, new antituberculosis treatment is urgent and imperative.

The discovery and clinical evaluation of a novel compound often takes years or even decades, yet the demand of new anti-TB drugs is immediate.

One option would be modifying the existing treatment of the antimicrobial agents to serve as new mycobactericidal drugs. Historically, M. tuberculosis was considered insusceptible to -lactam antibiotics due to the production of -lactamase (Finch, 1986). Nonetheless, combination of -lactam antibiotics and -lactamase inhibitors appeared to be active in vitro with latent clinical applications (Casal et al., 1987; Chambers et al., 1995; Cynamon and Palmer, 1983; Davies Forsman et al., 2015; England et al., 2012; Hugonnet et al., 2009; Tiberi et al., 2016).

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1.2 -Lactam Antibiotics

The first -lactam antibiotic, penicillin, was discovered in 1929 by Sir Alexander Fleming (Fleming, 1929). Since it was made clinically available in late 1940, -lactam antibiotics have been widely used as antibacterial agent, dominating the global market by over$20.6 billion in 2015, constituting over half of the systemic antibiotics market (Maliwal, 2016). -lactam antibiotics is certainly the most important and common antibiotics prescribed.

-lactam antibiotics disrupt bacterial cell wall synthesis by inhibiting penicillin-binding proteins (PBPs). PBPs are enzymes that facilitate the formation of peptidoglycan layer through catalysing D-alanine carboxypeptidase, peptidoglycan transpeptidase, and peptidoglycan endopeptidase (Spratt, 1977). Failure in peptidoglycan synthesis is an overture to irregularities in cell wall structure, leading to cell death from lysis (Page, 1984). PBPs bind to -lactam antibiotics due to their structural analogy to d-alanyl-d-alanine (Figure 1), the terminal amino acid residues of the nascent peptidoglycan (Nguyen-Dist`eche et al., 1982). The antibiotics irreversibly bind to residue Ser-403 of the PBPs active site and deactivate the PBPs (Fisher et al., 2005; Waxman et al., 1980). The signature core structure of -lactam antibiotics that facilitates their binding is the -lactam ring, a four-membered cyclic amide which the nitrogen bonds to the -carbon. Di↵erent families of -lactam antibiotics, classified by their -lactam nuclei (Table 1), have been expeditiously and systemically isolated and synthesised from 1940s through the 1980s (Boucher et al., 2013;

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Papp-Wallace et al., 2011; Fuchs et al., 1988; Birnbaum et al., 1985). The extensive clinical usage of -lactam antibiotics, with the rapid development of vaccination, have predominantly reduced the mortality of infectious disease by over 95% during the 19th century (Armstrong et al., 1999).

O

N

O

OH H S

N O R

O

NH

O

OH H

N O R

Penicillin d-alanyl-d-alanine

Figure 1. The mimicry of -lactam antibiotics to d-alanyl-d-alanine facilitates their binding to the active site of PBPs.

However, due to rising scientific, regulatory, and economic hurdles following the explosive antibiotics development, research and development on antibiotics shrank in the 1990s as a result of the egression of many pharmaceutical firms (Spellberg, 2014). From 1983, the number of new FDA approved antibacterial agents dwindled for 56% in 20 years (Spellberg et al., 2004). The growing resistance to antibiotics aggravated the situation.

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Table 1. Common -lactam antibiotics

Class Structure Examples

Pencillins

O N

H S N O

R¹C ampicillin

amoxicillin oxacillin penicillin G penicillin V

Cephalosporins

O N

COOH R² H S

N O

R¹C First generation:

cephaloridine, cephalothin Second generation:

cefoxitin, cefuroxime Third generation:

cefotaxime, ceftriaxone, ceftazidime

Carbapenems

O N

COOH R¹

OH meropenem

imipenem ertapenem doripenem

Monobactams

O SO₃H R² H N O R¹C

aztreonam

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1.3 -Lactamase and -Lactam Antibiotics Resistance

The problem of antibiotic resistance has casted the shadow over the e↵ectiveness of -lactam antibiotics from the very beginning. The mechanism of -lactam resistance was the first to be understood amongst antibiotics resistance. Abraham and Chain (1940) found penicillinase enzyme, a -lactamase, in the Penicillium cultures and in the extract of E. coli in the early small-scale production of penicillin.

Bacteria e↵ectively counteract the drug by expressing hydrolytic enzyme -lactamase to degrade the four-membered -lactam ring. Reports of penicillin-resistant staphylococci in patients emerged within a year of introduction (Rammelkamp and Maxon, 1942). Although the problem was first controlled by increasing the dosage, resistance quickly escalated to worldwide multiple-resistance (Finland et al., 1950; Finland, 1955).

Nowadays, it is not uncommon for an individual bacteria to possess multiple resistance genes for di↵erent antibiotics (King et al., 2017). The extensive use of antibiotics promoted the evolution of -lactamase, and to tackle the issue novel -lactam antibacterial agents were introduced, imposing an even more tremendous selective pressure on bacteria for resistance, wherefore spiral down a vicious cycle. World Health Organisation reported that antimicrobial medicines are available without a prescription in 60% of the participated countries (World Health Organization, 2015b). Clinical, industrial, and agricultural misuse and overuse of antibiotics have exacerbated the situation and diminished the e↵ect of antibiotics. It was estimated in 2014 that

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700,000 people die every year from drug-resistant strains of common bacterial infection, HIV, TB, and malaria (O’Neill, 2014), of which 200,000 from multidrug-resistant or extensively drug-resistant TB (World Health Organization, 2015a).

With over 4000 unique enzymes identified (Naas et al., 2017), many had attempted to categorise the -lactamase in order to understand and hence vanquish them. Jack and Richmond (1970) first classified the -lactamases by their functionality in 1970; Sykes and Matthew (1976) expended the compilation with other biochemical characteristics. Bush later proposed a reorganisation of the existing classification for both chromosomal and plasmid-mediated enzymes (Bush, 1989; Bush and Jacoby, 2010). However, Ambler classification is currently the most widely used (Ambler, 1980; Hall and Barlow, 2005). -lactamase is classified into four classes (A, B, C, and D) according to their nucleotide and amino acid sequences. Ambler classification is summarised in Table 2. Class A -lactamase, which is also referred to as penicillinase for its exceptional ability in penicillin hydrolysis, is the most abundant (Bush and Fisher, 2011). They are highly conserved in sequence and structure. The sequence alignment in Appendix A shows that the active site Ser-70 is conserved in all class A -lactamase, whilst another highly conserved residue Lys-73 with a basic side chain activates the hydrolysis (Drawz and Bonomo, 2010). Most class A -lactamases were driven by the increasing evolution pressure to develop resistance to cephalosporins and carbapenems to become extended-spectrum

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-lactamases (ESBLs) (Paterson and Bonomo, 2005), with the exception of a few broad-spectrum -lactamases (Sauvage et al., 2006).

Table 2. Ambler classification of -lactamase

Class Active site Subclass Enzyme type Examples

A

Serine

Broad spectrum TEM-1, TEM-2, SHV-1 Extended spectrum TEM-3, SHV-2,

CTX-M, GES-1

Carbapenemase KPC-1, KPC-2, KPC-3, SME, GES-2, IMI-1

C AmpC cephamycinase AmpC, CMY-2, P99,

ACT-1, FOX-1, MIR-1 Broad spectrum

D Extended spectrum OXA family

Carbapenemase

B1 VIM-1, IMP-1, NDM-1

B Metallo B2 Carbapenemases CphA

B3 FEZ-1

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1.4 BlaC -Lactamase

Amongst the M. tuberculosis -lactamases, BlaC, a broad-spectrum Ambler class A -lactamases, was identified as the most prominent (Hackbarth et al., 1997). Its structure and enzymology had been fully defined in the past decades (Voladri et al., 1998; Wang et al., 2006). Like other -lactamases, BlaC is considered a fully efficient enzyme and there is no rate determining step, but is only limited by di↵usion (Christensen et al., 1990; Hugonnet and Blanchard, 2007). The hydrolysis mechanism of -lactamase on -lactam antibiotics can be concluded in Figure 2 using ampicillin as an example. Similar to other class A -lactamase, the hydrogen bond network of BlaC -lactamase at the active site stabilises the substrate during hydrolysis. The hydrogen bond network is constructed by Ser-70 and Lys-73 on the ↵-helix H2; Lys-234, Thr-235, and Thr-237 on -strand B3;

Ser-130 and Gly-132 on the loop between H5 and H6; and Glu-166 on the ! loop (Figure 3). Ser-70 and Glu-166 also form indirect hydrogen bonds with the substrate through a highly conserved water molecule, which is involved in the deacylation step (Figure 4). BlaC exhibits a broad range of specificity, revealed from its comparable activity on penicillins and cephalosporins, possibly due to its flexible and sizeable substrate-binding pocket and its unique Gly-132 residue at the highly conserved Asn-132 of the SDN motif in penicillin binding proteins and -lactamases (Figure 5) (Sauvage et al., 2006; Wang et al., 2006).

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Figure2:Reactionmechanismof-lactamaseonampicillin.Theshadedpocketrepresentsthebindingpocketof-lactamase.The hydrolysisisinitiatedbynucleophilicattackbythehydroxylgrouponthesidechainofSer-70totheelectrophiliccarbononthe-lactam ring,whiletheamineonanearbyLys-234stabilisesthesubstrate(Knowles,1985;Christensenetal.,1990).

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Figure 3: Hydrogen bond network of the active site Ser-70 of acylated BlaC -lactamase. Lys-234, Thr-235, Thr-237, Lys-73, Ser-130, and Glu-166 form direct hydrogen bonds with the -lactam substrate ampicillin (green) (Wang et al., 2006).

Figure 4: Ser-70 is deacylated from the hydrolysed -lactam substrate with the aid of the hydrogen bonds formed by the side chain oxygen of Glu-166 via a water molecule. (Wang et al., 2006).

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(a) Acyl-enzyme intermediate of ampicillin (cyan) and Ser-70 (blue).

(b) BlaC -lactamase has a flexible and sizeable substrate-binding pocket. Cyan:

ampicillin.

Figure 5: Structure of BlaC -lactamase with ampicillin as substrate.

The structure was built using Crystallographic Object-Oriented Toolkit (Coot) (Emsley et al., 2010) and PyMOL (Schr¨odinger, New York, USA) based on the reported structures by Feiler et al. (2013) (PDB ID: 3ZHH) and Tremblay and Blanchard (2011) (PDB ID: 3N8L).

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1.5 -Lactamase-based Biosensor

The high affinity and enzyme specificity makes -lactamase a perfect candidate for -lactam antibiotics and inhibitors detection. Several approaches have been explored and employed. Gustavsson et al. (2002) developed an optical biosensor assay by measuring the surface plasmon resonance after immobilising the carboxypeptidase from Streptomyces R39 on the sensor surface covered with H1, a small organic molecule. Xiao et al.

(2016) constructed a photonic crystal-based -lactamase biosensor to detect the pH change in microenvironment during hydrolysis using icolloidal crystal hydrogel film with immobilised -lactamase. Prado et al. (2015) built an electrochemical biosensor with amperometric transduction with -lactamase on modified carbon paste. Another -lactamase-based electrochemical biosensor was designed by Vandevenne et al. (2018) by combining with hybrid -lactamase technology. Instead of immobilising or hybridising the -lactamase, Chan et al. (2008) labelled the protein with fluorescent probe in combination with site-directed mutagenesis. The biosensor remains as free enzymes for antibiotics detection and inhibitors screening (Chan et al., 2004, 2008).

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1.6 Site-Specific Labelling via Cysteines

Site-specific covalent labelling of proteins is an important tool in protein science. Fluorescent probes visualise protein interaction and translocation in real-time, as well as providing conformational and structural information of the protein (Fern´andez-Su´arez and Ting, 2008; Marks and Nolan, 2006;

Toseland, 2013; Tsien, 1998). Spin probes provide information on local dynamics and structure in combination with electron spin resonance (EPR) (Klare, 2013; Roser et al., 2016; Sahu and Lorigan, 2018). Amongst amino acids, cysteine is very popular for site-specific labelling due to its reactive thiol side chain and its relative rarity. Highly specific and efficient thiol-reactive probes, for example iodoacetamides and maleimides, that form thioether bonds with cysteine are common linkers to attach the probes to the protein (Clarke and Khalid, 2015). Introduction of cysteine at designated site is achieved by site-directed mutagenesis.

Site-directed mutagenesis modifies the DNA sequence of a gene to impose specific changes in the protein sequence. First successful site-directed mutagenesis in DNA by Weissmann's group induced an AT to GC transition using DNA polymerase I and N⁴-hydroxydCTP in 1974 (M¨uller et al., 1978).

However, the types of possible mutation are very limited and the efficiency is far from ideal as the specificity is not sufficient. Some improvements were developed, including Kunkel's method and cassette mutagenesis. Kunkel's method was suggested in 1985 by Dr. Thomas Kunkel. The unmutated DNA fragment is inserted into a phagemid, which is transformed into

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strain deficient in dUTPase and uracil deglycosidase. Accumulation of dUTP in cell due to the deficient in dUTPase increases incorporation of uracil into DNA. These single-strand DNA containing uracil are selected as template for mutagenesis. The need of selection is reduced by destroying the unmutated strands with uracil deglycosidase, leaving all the resulting DNA made up of mutated strands (Kunkel, 1985). The cassette mutagenesis uses a fragment of DNA instead of a DNA primer extended by DNA polymerase (Wells and Estell, 1988). The fragment is inserted into the plasmid directly with complementary sticky ends. Although the efficiency is sufficiently high, the major drawback of this method is the availability of suitable restriction recognition sites. Advancement in polymerase chain reaction (PCR) cleared a hurdle in site-directed mutagenesis by removing the restriction site limitation (Kleppe et al., 1971). PCR allows longer fragments to be built from deoxynucleoside triphosphates (dNTPs) with DNA polymerase based on a pair of complementary DNA primers. The amplified fragments contain the mutation and restriction sites on both ends.

The mutated protein produced is then labelled with fluorescent probes.

The fluorophore, however, may alter the secondary structure of the protein and hence its activity and properties. X-ray crystallography is frequently used to study the protein structure whilst molecular dynamic simulation is employed to predict and understand the the mechanism of the protein.

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1.7 Molecular Dynamics Simulation

Molecular dynamics (MD) simulation is a computational technique which studies biochemical process at atomic level. It furthers our understanding of protein from static structures obtained from experimental data, such as with X-ray di↵raction or nuclear magnetic resonance (NMR) spectrometry, by calculating a trajectory of all the atoms in the system under a set condition over time, thus allows the atomic fluctuations and conformational changes to be investigated.

There are two major approaches in MD simulations regarding the representation of the system: the ‘classical’ mechanics model and the

‘quantum’ or ‘first principles’ model. The classical mechanics approach considers the atoms as soft balls and bonds as elastic sticks, resembling a ‘ball and stick’ model. The dynamics of the system is described by the laws of classical mechanics. The ‘quantum’ or ‘first-principles’ MD is an advancement of the classical MD by including the quantum knowledge of the atoms and bonds. Car and Parrinello (1985) developed the method in 1980s. Bonding in the system is described by the electron density function for the valence electrons, whilst the dynamics of the ions, with only inner electrons and nuclei, is defined as in the classical model. However, due to the complexity of the quantum MD, extremely high-performance computer is required for the simulations of biomolecular systems which consists of tens of thousands of atoms in a timescale of nanoseconds.

The beginning of molecular dynamics simulation traces back to the

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late 1950s, the first simulation of the dynamics of liquids by Alder and Wainwright (1957). Rahman (1964) soon improved MD to create a realistic potential for liquid argon. Rahman and Stillinger (1971) then adopted the method in the MD simulation of a realistic system of liquid water. It was not until the late 1970s that MD simulations of protein were first performed by Karplus and his group (McCammon et al., 1977). Their work on the dynamics of a folded protein through solving the equations of motions was lauded with the Nobel Prize in Chemistry in 2013. Since the first calculation of protein motions based on classical dynamics, MD simulations has been widely exploited in the study of protein stability and folding problems (Arkun and Gur, 2012; Chodera et al., 2006; Khan et al., 2016; Miao et al., 2015; Pikkemaat et al., 2002), their impact on the motions and binding (Cote et al., 2017; Djuranovic and Hartmann, 2005; Dvorsky et al., 2000;

Freed et al., 2011), and even the e↵ect on their catalytic behaviours (Osuna et al., 2015; Warshel and Bora, 2016). MD simulations unveil information beyond the single crystal structures determined by X-ray crystallography and give details in the dynamic interactions between protein and its surrounding environment.

1.7.1 Main Principles and Approximations

MD simulations study the structural fluctuation at an equilibrated molecular system by generating the trajectory for all atoms in a system under a set of specified conditions and boundaries by solving the di↵erential

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equation of Newton's second law:

F = ma (1.1)

Fi

mi

= d²ri(t)

dt² (1.2)

where F is the force acting on particle i with mass m and displacment r_i(t) = (x_i(t), y_i(t), z_i(t)) at time t.

The force acting on the particle can only be calculated by integrating equation 1.2 when both the initial positions and velocities are known. The positions and velocities are then propagated over time using numerical integrators such as the Verlet algorithm (Verlet, 1967) to map a trajectory.

1.7.2 Initial Conditions

A thermodynamic ensemble characterises the possible microscopic states of a system with fixed total energy. The most common ensembles are microcanonical (NVE) ensemble (fixed number of atoms, N, volume, V, and total energy, E), canonical (NVT) ensemble (fixed number of atoms, N, volume, V, and temperature, T), grand canonical (µVT) ensemble (fixed chemical potential, µ, volume, V, and temperature, T), Gibbs (NPT) ensemble (fixed number of atoms, N, pressure, P, and temperature, T), and enthalpy (NPH) ensemble (fixed number of atoms, N, pressure, P, and enthalpy, H).

For ensembles with constant temperature or pressure, various approaches

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are available to model them in a realistic way. Thermostats control the “temperature”of the system by maintaining a constant total kinetic energy. The thermostat algorithms include Berendsen thermostat, Andersen thermostat, Nosé-Hoover thermostat, and Langevin dynamics. Berendsen thermostat controls the temperature of the simulation by weakly coupling with a heat bath to re-scale the velocities of particles (Berendsen et al., 1984). Although this violates the canonical ensemble and does not produce a correct trajectory with small systems, large systems consist of thousands of atoms benefits from its efficiency, and as the simulations are roughly correct, Berendsen thermostat is usually used in initial equilibration (Morishita, 2000). Anderdsen thermostat re-scales the velocities of particles by applying random collisions according to Boltzmann distribution at the desired temperature (Andersen, 1980). Nosé-Hoover thermostat is considered one of the most accurate method for constant temperature simulation (Hoover, 1985; Nosé, 1984a,b). It includes the heat bath explicitly as one imaginary particle for an extra degree of freedom in a Hamiltonian.

This approach achieves a more realistic canonical ensemble and is widely used in simulations. Langevin method simulates interactions with a solvent but it does not fully model an implicit solvent (Langevin, Paul, 1908; Lemons and Gythiel, 1997). It controls the temperature of the system by balancing a small friction term with random force selected from a Gaussian distribution.

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1.7.3 Force Field

The atomic interactions in the molecular system is described by a force field, where the interatomic forces are specified by potential U as a function of the atomic positions R.

Fi = rU(R) (1.3)

The potential U is composed of bonded and non-bonded energy terms:

U = Ubonded+ Unonbonded (1.4a)

where Ubonded and Unonbonded consist of the following terms respectively:

Ubonded = Ubond+ Uangle+ Udihedral+ Uimproper+ UU B+ UCM AP (1.4b)

Unonbonded= ULJ+ Uelec (1.4c)

The potential is therefore described by the energy function (MacKerell

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et al., 1998):

U = X

bonds

kb(b b0)²+ X

angles

k✓(✓ ✓0)²

+ X

dihedrals

k [1 + cos(n )] + X

impropers

k!(! !0)²

+ X

U rey Bradley

k_u(u u₀)²+ X

residue

u_{CM AP}⇣ , ⌘

+ X

nonbonded

"

✓Rminij

r_ij

◆12 ✓ Rminij

r_ij

◆6◆

+qiqj

"r_ij (1.5)

The first six terms are the bonded energy terms, whilst the last two terms are the non-bonded energy terms. The first two terms in the equation are for the bond stretching and bond angles, where kb and k✓ are the force constants, b b0 is the distance from equilibrium of that atom, and ✓ ✓0

is the angle from equilibrium between three bonded atoms. They prevent chemical changes such as bond breaking during the simulation and maintain the correct chemical structure. The third term in the equation is for the dihedrals (torsion angles) where k is the dihedral force constant, n is the multiplicity of the function, is the dihedral angle, and is the phase shift. The fourth term accounts for the improper angles where k! is the force constant and ! !0 is the out of plane angle. Improper angles are “virtual”torsion angles that define the chiral and planar centres. The fifth term is for the Urey-Bradley interactions, a cross-term accounting for angle bending using 1,3 non-bonded interactions, where ku is the respective force constant and u is the distance between the 1,3 atoms in the harmonic

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potential. The sixth term, CMAP, is a recent advancement to the force field (MacKerell et al., 2004) to improve the conformational properties of protein backbones. It is a cross-term for the protein backbone , values by grid-based energy correction maps. The non-bonded interactions between pairs of atoms (i, j) are the van der Waals force and electrostatic energy. The van der Waals energy is given by the Lennard-Jones potential, in which the short range repulsion is described by the r¹² term whilst the long range attraction is described by the r⁶ term. The last term, electrostatic energy, is calculated with the Coulombic potential where q is partial charge, " is the e↵ective dielectric constant, and rij is the distance between atoms i and j. All the parameters of the above terms have to be specified in the force field for every atom in the system. For novel compounds which are not included in the force field, parameterisation is necessary. Classical molecular mechanics (MM), semi-empirical quantum mechanical (SQM) and density functional theory (DFT) based methods are three common methods for novel parameterisation. MM assumes all atoms as classical particles based on the Born-Oppenheimer approximation and the potentials are calculated from semi-empirical relation. Whilst these assumptions allow calculations on larger molecules to be more feasible, excluding the quantum e↵ects leads to less accurate potentials. In contrast, DFT determines the properties of a many-electron system using functionals, the electron density, which is a function of space and time. It is possible to predict the material behaviour on the basis of quantum mechanical

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considerations, but this is much more computationally intensive compared to MM. SQM methods simplify Hartree-Fock theory by omitting some integrals based on experimental data, such as ionisation energies of atoms and dipole moments of molecules. SQM approaches showed similar performances on protein-ligand complexes as DFT methods (Yilmazer and Korth, 2013).

1.7.4 Long-Range Interactions Calculation

Although computational cost for long-range calculation has considerably reduced, the non-bonded interactions calculation is complicated and time-consuming. Therefore, di↵erent cut-o↵s are introduced to simplify the process while maintaining realistic conditions. Ewald summation calculates long-range interactions using a Fourier transform of the potential and the charge density (Ewald, 1921; Frenkel and Smit, 2002). Particle-mesh Ewald (PME) replaces direct summation of interaction energies between point particles with direct summation in real space for short-ranged part and summation in Fourier space for the long-ranged part (Darden et al., 1993;

Sagui and Darden, 1999). The method allows rapid convergence in energy and is fast and accurate. However, PME only works under a periodic boundary condition due to the assumption in Ewald summation.

1.7.5 Boundary Conditions and Solvent Treatment

Boundary conditions in MD simulations have to be carefully treated to avoid artifacts. The simulation box size is chosen to include all particles in the system. Instead of a set of fixed values at the edge, periodic conditions

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are used more often to mimic a bulk phase.

For simulation in solvent, the solvent model has to be decided between Generalised Born implicit solvent model and explicit solvent model. Water is represented by all-atom force field models in explicit solvent model, whilst the Generalised Born implicit solvent model also considers solvation energies and forces on biomolecules. Explicit solvent model is simplest and most widely used. There are several explicit solvent model, including SPC, SPC/E, and TIP3P (Jorgensen and Tirado-Rives, 2005).

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1.8 Aims of Study

In view of the increasing antimicrobial resistance in TB, the need for new mycobactericidal drugs is pressing. The combination of -lactam antibiotics and -lactamase inhibitor has pointed to new direction in mycobactericidal drug development. Current experimental treatments rely heavily on clavulanate as -lactamase inhibitor, but the emergence of clavulanate resistance overshadows the developing treatment (Egesborg et al., 2015; Soroka et al., 2015). New inhibitors are required to be used in place of clavulanate. In this report, the high functioning -lactamase, BlaC, of Mycobacterium tuberculosis was adopted and designed to be a sensitive and fast biosensor for screening the potential inhibitors for antituberculosis treatment along with -lactam antibiotics. Thr-216, a non-catalytic residue located at the edge of the binding pocket, was mutated into Cys (T216C), and a fluorophore was labelled on the mutated residue to undergo di↵erent fluorescence studies to examine its ability in working as a bioseneor.

Combination of electrospray ionisation mass spectrometry (ESI-MS) and fluorescence spectroscopy was used to confirm the hypothetic relationship between binding and fluorescence change. Kinetic studies were conducted to compare the activity of the mutated and labelled enzyme. The local dynamics of the label site was studied using site-directed spin labelling. The di↵erent hypotheses proposed were tested in molecular dynamics simulations to further understand our biosensor and optimise future -lactamase-based biosensor design.

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2 Materials and Methodology

2.1 Materials

2.1.1 Bacterial Strains and Plasmids

The class A -lactamase used was from Mycobacterium tuberculosis BlaC gene. The recipient strain of recombinant plasmid and for plasmid amplification was E. coli TOP10, and the host for protein over-expression was E. coli BL21 (DE3). Plasmid pRset-K, a kanamycin-resistant vector modified from pRset-A (Invitrogen), was employed for the production of -lactamase mutant in E. coli expression system. Ampicillin-resistant plasmid pRset-A had to be modified because the class A -lactamase expressed rapidly degrades ampicillin. Kanamycin resistance marker has to be introduced for selection. The plasmid map is shown in Figure 6.

2.1.2 DNA Manipulation Reagents

PfuUltra High-Fidelity DNA Polymerase (Stratagene, California, USA) was used in PCR reaction and site-directed mutagenesis. The primers were ordered from Invitrogen (California, USA).

2.1.3 Media

LB was purchased from A↵ymetrix (California, USA); sodium chloride was obtained from Sigma-Aldrich (Missouri, USA). Nutrient agar, tryptone, and yeast extract were purchased from Oxoid Ltd. (Basingstoke,

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Figure 6: Plasmid map of expression vector pRset-K. The BlaC gene was cloned between the Ndel and Hind III sites of the plasmid. Six codons of the 6xHis-tag were added before the 5’ end of the Hind III site of pRset-K. KanR represents the kanamycin resistant marker.

Hampshire, UK). The nutrient agar plates were prepared from sterilised 2.8%

w/v nutrient agar powder in double-deionised water containing 50µg/ml kanamycin. The LB medium used in pre-culture was prepared by dissolving 20 mg/L in double-deionised water and sterilising at 121 C . The 2 ⇥ TY medium was composed of 10 g of yeast extract, 16 g of tryptone, and 5 g of sodium chloride dissolved in 1 L of double-deionised water and was autoclaved for sterilisation.

2.1.4 Chemicals

All chemicals used were purchased and used without further purification or modification. Penicillin G, ampicillin, cefotaxine, cefoxitin, ceftazidine, ceftriaxone, cefuroxime, cephalothin, meropenem impenem, kanamycin, tazobactam, sulbactam, potassium clavulanate,

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3-(2-Iodoacetamido)-PROXYL were obtained from Sigma-Aldrich (Missouri, USA). Ammonium acetate, sodium phosphate monobasic monohydrate, sodium phosphate dibasic heptahydrate, potassium phosphate monobasic anhydrous, and potassium phosphate dibasic anhydrous were purchased from BD Medical (New Jersey, USA). Fluorescein-5-maleimide (F5M), 6-Iodoacetamidofluorescein (6-IAF), tetramethylrhodamine-5-maleimide, and 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) were obtained from ThermoFisher Scientific (Massachusetts, USA).

Isopropylthiogalactoside (IPTG) was bought from USB (Ohio, USA).

2.2 DNA Manipulation

2.2.1 Subcloning of the BlaC Mutant Gene into Expression Vector

The BlaC mutant gene was cloned between the Ndel and Hind III sites of the pRset-K plasmid by polymerase chain reaction (PCR) using Applied Biosystems Veriti Thermal Cycler (Foster City, California, USA). 0.5µl plasmid and 0.5µl pair of synthetic oligonucleotide primers (5’ →3’ forward primer and 3’ →5’ reverse primer) with nucleotide sequence of the BlaC -lactamase followed by 6 histidine codons were mixed with 5µl Taq DNA Polymerase PCR Bu↵er (Invitrogen) in 18µl Milli-Q water. 0.5 µl dNTPs was added with digestion protein to the mixture. The subcloning was conducted by Dr. H. K. Yap.

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2.3 Preparation and Transformation

2.3.1 Preparation of E. coli Competent Cells

E. coli TOP10 and BL21 (DE3) strains were cultured in 20 ml sterilised LB medium respectively at 37 C with shaking at 250 rpm until the optical density of the culture at 600 nm (OD₆₀₀) reached 0.4. The cells were harvested by centrifugation using Beckman Coulter Allegra X-22R Centrifuge (California, USA) at 4,000 rpm for 20 mins at 4 C. The cell pellets were then resuspended respectively in 10 ml of 100 mM CaCl₂ and kept in ice for 30 min. The cells were then collected again by centrifugation at 4,000 rpm for 30 mins at 4 C. The cell pellets were resuspended in 500 ml of 100 mM CaCl₂, before adding 50% sterilised glycerol to reach a final glycerol concentration of 15%. The mixtures were frozen in liquid nitrogen and immediately stored at 80 C.

2.3.2 Transformation of Competent Cells

The mixture of 2µl of the plasmid cloned with mutant gene and 100 µl E. coli TOP10 competent cell was incubated on ice for 30 min prior to heat shock at 42 C for 2 min. The mixture was then iced for 2 min. 200µl LB medium was added to the mixture, which was incubated at 37 C for 90 min with shaking at 300 rpm. The competent cells were streaked on a nutrient agar plate with 50µg/ml kanamycin, and was incubated at 37 C overnight.

Single colonies of E. coli TOP10 were picked and incubated in 5 ml LB

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medium with 50µg/ml kanamycin in a 50 ml conical tube at 37 C with shaking at 250 rpm overnight. 1 ml of each culture was then sent to BGI Genomics (Beijing Genomics Institute, China) for DNA sequencing. Cell cultures containing the -lactamase mutant gene were cultured to extract the plasmid using QIAprep Spin Miniprep Kit (Hilden, Germany). The plasmid was transformed to E. coli BL21 competent cell by heat shock at 42 C of iced competent cell and mutant gene containing plasmid mixture followed by incubation at 37 C for 90 min with shaking at 300 rpm in 200µl LB medium.

2.4 Expression and Purification

2.4.1 Expression of -Lactamase Mutants in E. coli

The -lactamase mutants T216C used in the study were overexpressed in E. coli BL21. The E. coli cells transferred with the -lactamase gene containing vector was streaked on a nutrient agar plate with 50µg/ml kanamycin, and was incubated at 37 C overnight. Single colonies of E. coli BL21 (DE3) were inoculated into 5 ml LB medium with 50µg/ml kanamycin in a 50 ml conical tube, and were incubated at 37 C with shaking at 250 rpm for 16 h. To scale up the production, 2 ml of the pre-culture was then transferred to 200 ml 2⇥ TY medium with 50 µg/ml kanamycin. The culture was again incubated at 37 C with shaking at 250 rpm. In order to closely monitor the cell growth, the optical density of the culture at 600 nm (OD600) was measured regularly. Once the OD600 reached 0.6, 200µl of 0.2 M filtered

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isopropythiogalactoside (IPTG) was added to the 200 ml culture to induce the expression of the -lactamase mutants. The culture was incubated for an additional 4 h before it was collected. The culture was collected by centrifugation using Sorvall LYNX 6000 Superspeed Centrifuge (Sorvall, Connecticut, USA) at 10,000 rpm for 20 mins at 4 C . The cell pellets was collected and stored at 20 C.

2.4.2 Purification by Nickel Affinity Chromatography

Prior to the chromatographic purification, the intracellular -lactamase mutant has to be extracted by homogenisation. 20 ml of solubilisation bu↵er (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4) was added to the cell pellet from 200 ml culture, and the cell pellet was resuspended by shaking on a vortex mixer. Next, the suspended cells were lysed by CF1 High Pressure Cell Disrupter (Constant Systems, Daventry, UK) equipped with a cooling system to maintain the temperature at 4 C. The pressure was set at 20 Kpsi and the cells were lysed thrice. To remove the cell debris, the bacterial lysate was subjected to centrifugation at 10,000 rpm for 1 h at 4 C. The supernatant was collected and filtered using 0.45µm low protein binding filter (Pall, New York, USA) before loading into a nickel affinity column.

As all the -lactamase mutants have a 6⇥His-tag at the C-terminus, they can bind to the nickel affinity column. Purification was performed using an Amersham Pharmacia ¨AKTA Pure 25 FPLC System (Amersham

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Pharmacia Biotech Inc., California, USA). The column used was a 5 ml HisTrap™chelating HP column (GE Healthcare, Illinois, USA). The column had to be prepared before loading the protein sample. After the column was thoroughly washed with 20% ethanol and Milli-Q water, 0.1 M nickel(II) sulphate was injected into the column. Unbound nickel ions were washed out by Milli-Q water. Next, the nickel charged column were pre-equlibrated with start bu↵er (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4). The filtered supernatant from the previous step was loaded into the column at a speed of 5 ml/ min, and the column was washed with 10 column volumes (CV) of start bu↵er to ensure all the unbound protein was washed out. By increasing the concentration of imidazole gradually, the His-tagged proteins were eluted and collected. Di↵erent fractions were then analysed using SDS-PAGE to identify the protein and examine the purity. For long-term storage, bu↵er exchange to 50 mM potassium phosphate (pH 7.0) was performed on the fractions containing the target protein using Amicon Ultra-4 (NMWL=10,000) centrifugal filter device (Millipore, Billerica, USA) and stored at 20 C.

2.4.3 Purification by Gel Filtration Chromatography

Purification was conducted using an Amersham Pharmacia ¨AKTA Pure 25 FPLC System (Amersham Pharmacia Biotech Inc., California, USA) equipped with Superdex 200 10/300 GL size exclusion column (GE Healthcare, Illinois, USA). The column was first washed with 20% ethanol

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and Milli-Q water and equilibrated with running bu↵er (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4). The labelled protein was concentrated to less than 1 ml and filtered with 0.45µm low protein binding filter (Pall, New York, USA) before loading into the gel filtration column.

The flow rate was set at 0.5 ml/ min and the pressure was monitored at below 1.5 MPa. Di↵erent fractions were collected and then analysed using SDS-PAGE to examine the purity. Bu↵er exchange to 50 mM potassium phosphate (pH 7.0) was performed on the fractions containing the pure labelled protein using Amicon Ultra-4 (NMWL=10,000) centrifugal filter device (Millipore, Billerica, USA) and stored at 20 C.

2.5 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis is an analytical technique to separate the components in a mixture, and to identify each component. In molecular biology, it is usually performed to examine the purity and mass of protein samples. As the protein samples run through the gel, di↵erent proteins are separated by their electrophoretic mobility.

12% SDS-PAGE in a Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories, California, USA) was used in the analysis. 10µl of the protein fractions were mixed with equal volume of SDS loading dye (0.5 M Tris-HCl (pH 6.8), 10% glucerol, 2% SDS, 10% -mercaptoethanol, 0.05% bromophenol blue) and the mixtures were boiled for 5 min before loading on the SDS-PAGE gel

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consisted of 5% stacking gel (pH 6.8) and 12% separating gel (pH 8.8). Low range standard, a mixture of six proteins with known masses, from Bio-Rad (California, USA) was used as a comparison, including phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin (45.0 kDa), bovine carbonic anhydrolase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg white lysozyme (14.4 kDa). Electrophorsis was performed in running bu↵er (25 mM Tris, 192 mM glucine, 1% SDS, pH 8.3) at 200 V for 60 min. After the electophorsis, the gel was stained with Coomassie Blue solution (10% acetic acid, 10% methanol, 0.01% Coomassie Blue R250) for 30 mins, followed by destaining with destain solution (10%

acetic acid, 10% methanol) with shaking until the protein bands were seen against a clear background.

2.6 Protein Concentration Determination

The protein concentration was determined using Bradford assay, bicinchoninic acid (BCA) protein assay, and spectrophotometry.

The Bradford assay was carried out in a total volume of 1000µl, 800µl protein sample diluted with double-deionised water and 200 µl Bradford reagent dye (Bio-Rad Laboratories, California, USA). The mixtures were then 10 min incubated at room temperature in order to form stable protein-dye complexes. The absorbance was measured using Ultrospec 3100 Pro UV/Visible Spectrophotometer (GE Healthcare / Amersham Biosciences, Buckinghamshire, UK) at the wavelength of

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595 nm. The protein concentration was calculated from the standard protein-concentration-absorbance curve, which was plotted from the absorbances of bovine serum albumin (BSA) (Sigma, Missouri, USA) in di↵erent concentrations, range from 1µg/ml to 10 µg/ml.

The BCA protein assay was performed using the Pierce BCA Protein Assay Kit (ThermoScientific, Rockford, USA). Firstly, 50 parts of BCA Reagent A and 1 part of BCA Reagent B were mixed together to give a working reagent. Similar to the Bradford assay, a standard protein-concentration-absorbance curve was plotted for comparison. The range of BCA concentrations was between 10µg/ml and µg/ml.

Protein in aqueous solution absorbs UV light with peptide backbone absorption maximum at 205 nm and aromatic rings side chain (tryptophan and tyrosine) absorption maximum at 280 nm (Perkins, 1986; Scopes, 1974).

The absorption at 280 nm is has a larger concentration range but relatively less sensitive than at 205 nm as the molar absorptivity is higher. As the absorption at 280 nm depends on the number of residues with aromatic rings in the protein, extinction coefficient " has to be calculated using ProtParam (Gasteiger et al., 2005). The protein concentration can then be calculated using Beer-Lambert Law

A = "c` (2.1)

where A is the absorbance, " is the molar absorbtivity coefficient (" = 30 035 M ¹cm ¹ at A280), c is the concentration of the solution, and ` is the path length. All absorbance measurements were

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conducted using NanoDrop™One Microvolume UV-Vis Spectrophotometer (ThermoScientific, Rockford, USA).

2.7 Labelling of -Lactamases with Fluorescent Dyes

Thiol-reactive fluorescent dye can be easily attached to any molecule having free thiols, such as cysteine side chain. For decades, it has been widely reported as an efficient label to protein and antibiodies to use as fluorescent probes. To label the protein in the native condition, 10-fold molar excess 0.2 M fluorescence dye in dimethylformamide (DMF) was added to the 1 mg/ml -lactamases in 50 mM potassium phosphate bu↵er (pH 7.0). The mixture was then incubated in the dark with shaking at 800 rpm at room temperature for 2 h. Afterwards, the excess dye was removed by bu↵er exchange to 50 mM potassium phosphate bu↵er (pH 7.0) for at least six times, using Amicon Ultra-4 (NMWL=10,000) centrifugal filter device (Millipore, Billerica, USA), until no dye can be washed out from the protein.

The labelled mutant was then frozen in liquid nitrogen, and stored at 20 C.

2.8 Labelling of -Lactamases with Spin Probe

Local dynamics of protein can be inspected by combination of site-directed spin probe labelling and EPR. The protein was labelled with 10-fold molar excess 0.2 M spin probe, 3-(2-iodoacetamido)-PROXYL, in DMF. The mixture reacted under the same conditions as described in 2.7.

The unreacted 3-(2-iodoacetamido)-PROXYL was removed in gel filtration

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chromatography, which details can be found in 2.4.3.

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3 Characterisation and Functional Studies of the BlaC-based Biosensor

3.1 Introduction

Class A and class C -lactamases have been modified through site-directed fluorophore labelling into fluorescent biosensors to detect for -lactam antibiotics in food (Chan et al., 2004, 2008; Cheong et al., 2014; Hu et al., 2016; Tsang et al., 2011, 2016). The fluorescence signals were prompted when a substrate or inhibitor bound to the protein active site and provoked the translocation of the fluorophore, changing its surrounding environment (Wong et al., 2011a,b). An extended-spectrum -lactamase (ESBL) BlaC, which is responsible for the -lactam antibiotic resistance in M. tuberculosis, was modified by substitution of residue Thr-216 to Cys and Cys-285 to Ser by site-directed mutagenesis and subsequent cysteine labelling with thiol-reactive fluorescence probes. Primary fluorescence spectra for mutants labelled with fluorescein-5-maleimide (T216Cf), 6-iodoacetamidofluorescein (T216Ciaf), 6-bromoacetyl-2-dimethylaminonaphthalene (T216Cb), and tetramethylrhodamine-5-maleimide (T216Cr) against penicillins were examined. After the most promising modified mutant T216Cf was chosen, its functionality against various -lactam antibiotics and -lactamase inhibitors was tested and compared with another modified mutant T216Ciaf.

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The relationship between fluorescence intensity change and inhibitor binding was further studied by monitoring the fluorescence signal using fluorescence spectrometry and the formation of enzyme-inhibitor complex using mass spectrometry simultaneously. A microplate reader equipped with fluorescence spectrometer was exploited for automated screening and fast detection, which showed the capacity of T216Cf in large-scale drug screening.

Kinetic studies revealed e↵ect of mutagenesis and flurophore labelling on the enzymatic function to ensure the T216Cf screening is indicative.

-Lactamase inhibitor protein (BLIP) was isolated from Streptomyces clavuligerus in 1994 (Strynadka et al., 1994). It strongly binds with a variety of class A -lactamase including TEM-1 (Wang et al., 2009; Zhang and Palzkill, 2004), SHV-1 (Hanes et al., 2011), and KPC-2 (Chow et al., 2016) through protein-protein interactions, hence is a potent inhibitor. The addition of BLIP to the BlaC -lactamase-based biosensor T216Cf induced an interesting fluorescence signal, evidenced for the first time the binding of BLIP to BlaC -lactamase.

3.2 Methods

3.2.1 Electrospray Ionisation Mass Spectrometry (ESI-MS) Studies of -Lactamase Mutants

Electrospray Ionisation Mass Spectrometry (ESI-MS) is a common technique adapted to study the mass and the purity of a protein sample.

Prior to the measurement, the bu↵er exchange of the protein samples

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was performed from 50 mM potassium phosphate bu↵er (pH 7.0) to 20 mM ammonium acetate (pH 7.0) for six times, using Amicon Ultra-4 (NMWL=10,000) centrifugal filter device (Millipore, Billerica, USA). 100µl of the protein was unfolded by mixing with equal volume of acetonitrile with 2% (1:1 v/v) formic acid. Nitrogen was used as the desolvation gas, the cone gas, and the nebulising gas. The scanning range of the mass spectrometer was set at m/z 600-2,000. The capillary voltage and the cone voltage were set at 3 kV and 30 V correspondingly. The rates of the desolvation gas and the cone gas were adjusted to 400 L/h and 50 L/h respectively, while the level of the nebulising gas was opened to maximum. The mass spectrometer was calibrated by sodium iodide. The protein mixture was injected into the mass spectrometer at a flow rate of 300µl/min, with the aid of a syringe pump. A multiply-charged mass spectrum, which contains multiply-charged protein ion peaks, was obtained. The average molecular mass of the protein was calculated from the multiply-charged mass spectrum by deconvolution using the MassLynx 4.1 Transformation Program (Micromass, Altrincham, Cheshire, UK). The ESI-MS measurements were conducted by Dr. Pui-kin So.

3.2.2 Continuous Wave Electron Paramagnetic Resonance (CW EPR)

CW EPR spectroscopy was performed on SPINSCAN X spectrometer (Adani Systems, Minsk, Belarus) fitted with a Q cavity. 1 ml purified mutant

Copyright Undertaking

DEVELOPMENT OF

-LACTAMASE-BASED BIOSENSOR FOR ANTI-TUBERCULOSIS DRUG

SCREENING

ZOE CHAN

PhD

The Hong Kong Polytechnic University

2019

The Hong Kong Polytechnic University

Department of Applied Biology and Chemical Technology

Development of -Lactamase-based Biosensor for Anti-tuberculosis Drug

Screening

Zoe Chan

A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy

August 2018

Certificate of Originality

Acknowledgements

Table of Contents

List of Figures

List of Tables

Abbreviations

1 Introduction

1.1 Mycobacterium tuberculosis

1.2 -Lactam Antibiotics

1.3 -Lactamase and -Lactam Antibiotics Resistance

1.4 BlaC -Lactamase

1.5 -Lactamase-based Biosensor

1.6 Site-Specific Labelling via Cysteines

1.7 Molecular Dynamics Simulation

1.8 Aims of Study

2 Materials and Methodology

2.1 Materials

2.2 DNA Manipulation

2.3 Preparation and Transformation

2.4 Expression and Purification

2.5 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2.6 Protein Concentration Determination

2.7 Labelling of -Lactamases with Fluorescent Dyes

2.8 Labelling of -Lactamases with Spin Probe

3 Characterisation and Functional Studies of the BlaC-based Biosensor

3.1 Introduction

3.2 Methods