1 GTG GCG GCA CGG ATC ACG ACA GAG CGC ATC ACC GAC CAT CCG GAC GCT GAC
M A A R I T T E R I T D H P D A D
52 GCC ATC ACC CTC CAG GGC GTC CTG GAC GCG CTG GTC GAT CCG GTG CGC CGC
A I T L Q G V L D A L V D P V R R
103 AGC ATC GTC CGG CAG CTG GCT AAG GCA CCC GAG GAC ATC GCC TGC GGC ACC
S I V R Q L A K A P E D I A C G T
154 TTC GAC ATC ACC GTC TCC CGC TCG ACC GGC ACT CAC CAC TTC AAG GTG TTG
F D I T V S R S T G T H H F K V L
205 CGC CAG GCC GGG ATC ATC AGG CAG TAC TAC ATC GGC ACC TCG AAG ATG AAC
R Q A G I I R Q Y Y I G T S K M N
256 ACG CTT CGC ACC GAT GAT CTC GAT CAG GCC TTC CCC GGC CTG CTC ACC GCG
T L R T D D L D Q A F P G L L T A
307 ATC GTC GAC GCC GCG GCC AGG GAG AGC TGA AAA AAA AAA AAA AAA AAA AAA
I V D A A A R E S - AAA AAA AAA AAA AAA AAA AAA
Figure 1.17: Genetic sequence and corresponding amino acid sequence for MmyJ [97].
pattern of suspected non-metal binding ArsR proteins containing theα2α4 motif. However, as this system is regulating Mm production, and is not proposed to involve metal sensing, it is possible that cysteine residues are not needed if MmA or one of its biosynthetic intermediates is sensed by MmyJ. If this is the case it would be the first reported instance of an ArsR family protein sensing a non-metallic ligand.
1.4
Project Aims & Objectives
The main aim of this project is to investigate MmyJ and determine its structure and function, and hence conclude whether it is truly a novel ArsR-like transcriptional repressor and is the first instance of an ArsR family protein known to bind an organic molecule, rather than a metallic ligand. An in depth bioinformatics analysis will be carried out, alongside experimental work with expressed protein and amplified fragments of the mmr-mmyJ intergenic region to determine binding characteristics. Work will also be carried out into identifying the binding ligand and/or conditions that cause dissociation of MmyJ from DNA if it is proven to be a transcription factor. Also, attempts will be made to confirm that the identified imperfect inverted repeat surrounding the mmyJ/mmr promoter regions is in fact the binding site of MmyJ, confirming its role as a transcriptional repressor of itself as well asmmr. Once the DNA
1.4 Project Aims & Objectives 1 INTRODUCTION
target region and ligand have been identified, surface plasmon resonance (SPR) will be used to determine the binding kinetics of the interactions.
As well as the above functional investigation, work will be carried out to determine MmyJ’s structure by solution and solid state NMR, as well as X-Ray crystallography if conditions can be determined that lead to crystallisation of the protein. From this, work will then be carried out, if possible, to observe the structure of MmyJ when bound to both DNA and its ligand, with the hope that direct comparisons between these structures and that of apo-MmyJ will lead to insights into the specific residues needed for each interaction. Data acquired through NMR will also be used to complement the analysis by SPR, investigating the link between residue mobility and binding kinetics at the interaction sites.
2 BIOINFORMATIC ANALYSIS OF MMYJ
2
Bioinformatic Analysis of MmyJ
Before characterising MmyJ by experimental means, bioinformatic studies were carried out to investigate MmyJ and determine its similarities and differences to other ArsR proteins.
2.1
MmyJ - Basic Properties
The amino acid sequence for MmyJ is shown in Figure 2.1. This was first determined by forming a cosmid library based on the linear plasmid SCP1 fromS. coelicolor A3(2), sequencing it and translating the coding sequences (CDS) from these cosmids. Specifically, cosmid C73 contains the entire methylenomycin pathway [99]. As can be seen, the resulting protein corresponding to the mmyJ gene is 111 amino acids long, with a corresponding mass of 12135.8 Da. The amino acid composition can be seen in Figure 2.1b. This sequence was then entered into the ProtParam [102] web tool in order to calculate basic physical properties of the protein, relevant for later work. One such property calculated was the extinction coefficient, found to be 2980±300 M−1cm−1, corresponding to an absorbance at 280 nm of 0.246±0.02 when at a concentration of 1 g/L.2 This allowed the concentration of MmyJ to be determined when produced in the laboratory (although a modification to 0.281±0.03 was required for recombinant MmyJ with histidine tag, as described in Section 3). Also, the computations indicated that
2Calculated uncertainty of 10% in approximation of extinction coefficient is due to lack of Trp residues [102].
10 20 30 40 50 60
MAARITTERI TDHPDADAIT LOGVLDALVD PVRRSIVROL AKAPEDIACG TFDITVSRST
70 80 90 100 110 h
GTHHFKVLRQ AGIIRQYYIG TSKMNTLRTD DLDQAFPGLL TAIVDAAARE S (a)
(b)
Figure 2.1: (a) Amino acid sequence and (b) amino acid composition of MmyJ, determined by coding sequence (CDS) analysis.