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Antibiotic resistance has increased significantly in the recent years, making treatment of bacterial infections harder. The development of antibiotic resistance in bacteria can be due to several factors. Antibiotic resistance can develop as an evolutionary process, based on the selection of bacteria with an enhanced ability to survive doses of antibiotics produced by other bacteria within the environment. This process has been accelerated by the over prescription and inaccurate use of antibiotics in medicine. This has contributed to an increase in the order of 10-5 per genome per generation in the adaptive mutations of E. coli.
Genes encoding antibiotic resistance may also be transferred from one bacterium to another and between different bacterial species. This occurs via horizontal gene transfer, which is the uptake of DNA or RNA between bacteria via the transfer of plasmids that posses genes encoding antibiotic resistance. Plasmids can possess different genes with diverse resistance mechanisms to unrelated antibiotics. Bacteria are able to possess resistance to multiple antibiotics simultaneously; this may result when a gene encoding a resistance mechanism is capable of providing resistance to more than one antibiotic. Uptake of DNA or RNA can also occur by transduction and transformation. Point mutations within the bacterial genome may also occur naturally during chromosomal replication, or induced when a virus gene is incorporated within the host genome causing genetic rearrangements.
As transference can occur between different bacterial species antibiotic resistance genes from E. coli can therefore be transferred to Staphylococcus aureus. This can lead to multidrug resistant bacteria such as MRSA (Methicillin Resistant Staphylococcus aureus). MRSA can lead to many people being hospitalized each year, after infection, some may die as a result.
There are four main molecular mechanisms that bacteria employ to inhibit antimicrobials; inactivation of the antimicrobial directly or inactivation via modification (e.g. ï¢ - lacatamases, enzymes that phosphorylate, adenylate, or acetylate aminoglycoside antibiotics), alteration of the antibiotic target site of action, alteration of the metabolic pathway affected, possession of an alternative metabolic pathway and bypassing the affected pathway. The accumulation of antimicrobials may also be reduced by increasing antibiotic active efflux (31). Proteins that cause active efflux of antibiotics will be the central point of my interest in my research.
Antibiotics are vital in the fight against bacterial infection. Antibiotics are characterised into narrow-spectrum and broad-spectrum antibiotics based on their target specificity. Narrow spectrum antibiotics target certain gram-negative or gram-positive bacteria, whereas broad spectrum antibiotics affect a wide variety of bacteria. Antibiotics work using various mechanisms of action. The majority of antibiotics target bacterial functions or growth processes. Antibiotics are also known to target a variety of other sites; cell wall (penicillins (Fig 1.7.)), cell membrane (polymixins) and interact with essential enzymes (quinolones). Antibiotics that target protein synthesis (e.g. aminoglycosides) are commonly bacteriostatic.
Figure 1.7. Chemical structure of penicillin (A) and chloramphenicol (B).
1.3.1. Bacteria Cell Structure
Bacteria can be classed in to two main groups; gram-positive and gram-negative. Classification of bacteria is made based on how they react to gram stain (crystal violet stain). Gram-positive bacteria have a thick cell wall made of peptidoglycan (Fig 1.1.) (50-90% of cell wall) that stains purple. Gram-negative bacteria have a thinner cell wall (10%) that stains pink. Gram-negative bacteria are also morphologically distinct from gram-positive bacteria due to the presence of the outer membrane, which contains lipids and is separated from the cell wall by the periplasmic space (Fig 1.1.).
Figure 1.1. Generalized structure of gram-positive and gram-negative bacteria, illustrating the difference in the bacterial composition.
1.3.2. Bacterial Outer membrane
The outer membrane is a double layered lipid membrane forming the outermost surface of the bacterium (Fig 1.2.). It encloses the peptidoglycan and inner membrane. The outer membrane is unique as the outer layer is primarily composed of lipopolysaccharide (LPS) (Fig 1.2.). The LPS layer provides an increased resistance to host-defence proteins, digestive enzymes and environmental toxic agents. The LPS has decreased permeability to hydrophobic and charged compounds. Decreased permeability is caused by limited mobility LPS molecules that are increased by the presence of ions (e.g. Mg2+ or Ca2+), which cause strong interactions between LPS molecules.
Porins are one of the main constituent proteins of the outer membrane in gram-negative and some gram-positive bacteria. Porins are beta barrel proteins that cross the outer membrane that form a pore allowing substances to passively diffuse through. Typical substances include small metabolites such as sugars, ions and amino acids. Other proteins of in the outer membrane include outer membrane factors (OMF) (e.g. tolC) that are involved in a variety of functions.
1.3.3. Structure of outer membrane porins
Porins are composed of beta sheets, which are usually connected by beta turns on the cytoplasmic plane and long loops of amino acids on the outer plane. A cylindrical tube called a beta barrel is formed by the antiparallel arrangement of beta sheets. The amino acids within beta sheets alternate between polar and non-polar residues. The non-polar residues face outwards to interact with the non-polar lipid membrane. The polar residues face into the pore allowing their interaction with the aqueous channel. Negatively charged amino acids line the majority of the porin channel. The arrangement of these amino acids on opposite sides of the channel creates a transversal electric field across the pore. A loop called the eyelet projects into the cavity of the porin channel, which results in the partial blockage of the channel. The eyelet defines the size of solute that can pass through the channel. The eyelet is generally found between strands 5 and 6 of each barrel. Two bound calcium atoms partially compensate for the eyelets negative charge.
A number of gram negative bacteria are known to express ompF, which contributes to the survival of bacteria under conditions of different osmolarity. The gene encoding ompF (mar gene) is upregulated under conditions of low osmolarity. OmpF is also known to play a role in bacterial drug resistance, as it increases the bacterial permeability to certain drugs by providing channels for these drugs to be transported into the bacteria (e.g. quinolones, tetracyclines, and β-lactams). The permeability of bacteria to certain drugs is reduced by the decreased expression of ompF, and it is suggested that this decreased expression of ompF is key in the development of multidrug resistant bacteria.
The ompF porin is a trimeric integral membrane protein, which allow for the transportation of small hydrophilic molecules such as waste products and nutrients through the outer membrane of E. coli. ompF monomers consists of 16 stranded antiparallel barrel. Three monomers interact strongly with one another to form a homotrimer. There are eight short turns on the periplasmic plane and eight long loops on the extracellular plane. The entrance of the barrel is partially blocked by six of the extracellular loops packed together (loops 1 and 4-8). Certain loops are involved key functions of ompF; loop 2 - monomer-monomer interactions and loop 3 - folds within the barrel restricting the size of the pore. A strong electrostatic field is present across the channel, which is generated by charged residues within the pore.
Figure Visualization of ompF topology in bacterial gram-negative outer membrane. The three protomers of ompF that form the homotrimer are shown in green, red and blue.
tolC is a component of the outer membrane and belongs to the OMF family. tolC is essential in the efflux of a variety of compounds; including small inhibitory molecules, antibacterial drugs and large proteins. Substrate specificity and access through the pore is regulated by the interaction of the substrate with a specific inner membrane translocase.
tolC is assembled as a trimer of a 428-residue protomer with the long axis measuring 140Å. Approximately 100Å the trimer body forms a cylinder with an internal diameter of 35Å. The distal end of the trimer is open, which allows for the access of wide solvents. The proximal end on the other hand is tapered and almost closed. This arrangement causes the large interior cavity of the trimer to be solvent-filled. The tolC trimer can be separated into three distinct domains; a ï¢ - barrel domain located within the membrane, a central mixed ï¡/ï¢ domain and a ï¡- helical domain located furthest away from the membrane (Koronakis et al., 2000). Each monomer's amino acid chain runs from the membrane down to the far end of the molecule four times. The ï¢ - barrel domain consists of 4 strands from each monomer, which joins antiparallel to form the 12 stranded ï¢ - barrel domain. The four strands of each monomer also join to form the main body of tolC a 12-stranded antiparallel barrel, ï¡- helical domain. The ï¢ - barrel domain has an anticlockwise twist deferring to the ï¡- helical domain which has a clockwise twist. A mechanism for the facilitation the direct transport of substrates across two membranes and periplasmic space is revealed by the structure of the tolC. The efflux of small molecules relies on the opening of the tapered proximal end in order to release the molecule. Alternatively the efflux of proteins requires the partial folding of the proximal end.
The proposed mechanism for transport of substrates through tolC suggests that the inner helix rotates around its adjoining helix, causing the pore to open. The two adjoining helices show significant sequence homology. The sequence homology allows for the shifting in structural conformation via an untwisting mechanism. The structural shift involved in the mechanism is triggered via interaction of tolC with a specific inner membrane protein. The interaction site may occur at the ï¡/ï¢ domain.
Figure Visualization of tolC topology in bacterial gram-negative inner membrane. The three protomers of tolC that form the homotrimer are shown in green, red and blue.
1.3.4. Bacterial Inner membrane
The inner membrane consists of a phospholipid bilayer (Fig 1.2.) and is the major physiological barrier between the outside of the cell and the cytoplasm, due to its inherent impermeability to many nutrients and waste products of metabolism. The inner membrane is therefore the site of various biochemical reactions (Fig 1.2.). Reactions are carried out by various proteins including the transport of substances into or out of the cytoplasm, transmission of signals into the cytoplasm and the generation of energy via the use of a proton motive force that is maintained by proteins in the inner membrane. Proteins within the inner membrane are also involved in cell division. 3 % - 15 % of genomic potential in organisms encodes for transport proteins.
Transportation is most commonly energy linked, allowing transportation against the electrochemical gradient. Drugs/antibiotics, which often are able to enter the membrane, can then be transported from the cell via integral membrane proteins (Fig 1.2.) thereby conferring resistance.
Figure 1.2. Representation of the gram-negative bacterial cell membranes. The location of various proteins within the cell membranes is shown and the interaction between the outer and inner membrane (cytoplasmic membrane) proteins and examples of their functions (Faraldo-Gomez and Sansom, 2003).
1.4.1. Classes of MDR efflux pumps
Multiple families of membrane proteins expressed within the inner membrane of gram-negative bacteria confer drug/antibiotic resistance. Proteins that confer drug resistance of two or more drugs are classed as multidrug resistance (MDR) transporters (Fig 1.3.). Multidrug resistance transporters can be classified into two major classes based on bioenergetic and structural criteria; adenosine 5'- triphosphate (ATP)-binding cassette (ABC) proteins and secondary multidrug transporters. ABC transporters directly coupling drug efflux to ATP hydrolysis (e.g. sav1866). Secondary multidrug transporters utilize the transmembrane electrochemical gradient to drive the extrusion of drugs. (Fig 1.3.) Secondary transporters include four families; resistance/ nodulation/ division superfamily (RND) (e.g. acrB, acrF, acrD, yhiV), multiple antimicrobial toxin extrusion (MATE) family (e.g. norM), small multidrug resistance (SMR) family (e.g. emrE, tehA), and the major facilitator superfamily (MFS) (e.g. emrD, mdfA, emrB) (Fig1.3.). These families of transporters are prevalent within microbial genomes. MDR transporters are diverse in there structure; which allows them to be diverse in their substrate specificity (1).
Figure 1.3. Families of inner membrane multidrug transporters. The seven families are; ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic-compound extrusion (MATE) family, the small multidrug resistance (SMR) family and the resistance nodulation division (RND) family. Examples of each family are shown with examples of known substrates of each.
MDR transporters are classified by a range of criteria; whether the pump has single or multiple components, the substrate type transported by the pump, the number of transmembrane-spanning regions and the pump's energy source. MDR transporters from multiple families and/ or multiple types from the same family can be expressed within a single organism. E. coli can express multiple types of Acr efflux pumps for example.
These efflux pumps consist of a transporter protein (e.g. acrB (acriflavine resistance protein B)) located in the bacterial inner membrane, an accessory protein (e.g. acrA) located in the periplasmic space and an outer-membrane protein (e.g. tolC) located in the outer membrane of the bacterium. Transport of substrate through the RND-family pumps is driven by the proton motive force, which is an electrochemical gradient created by the movement of hydrogen ions. Gram negative bacteria RND transporters form functional tripartite systems, which are involved in MDR (Fig 1.3.)
ABC Multidrug Transporters
There are two ATP binding cassettes in ABC multidrug transporters, which are driven by the free energy of ATP hydrolysis. All ATP-dependent drug efflux proteins known to date belong to the ABC superfamily. ABC transporters usually require four distinct domains; two hydrophilic nucleotide-binding domains containing the Walker A and B motifs and the ABC signature and two highly hydrophobic membrane domains each consisting of six transmembrane ï¡-helices. The majority of bacterial ABC drug transporters mediate the efflux of specific antibiotics. It has been shown that a structural change is required for substrate efflux by ABC transporters, which can be initiated via substrate binding and ATP hydrolysis.
Staphylococcus aureus expresses the ABC transporter sav1866. sav1866 is responsible for the transport of a wide range of structurally diverse cytotoxic drugs and endogenous lipids. Each monomer of sav1866 consists of 6 transmembrane helices. Two sav1866 monomers join to form a homodimer. sav1866 is 120 Å long, 65 Å wide and 55 Å deep. There are two domains within each subunit of sav1866; an amino-terminal transmembrane domain and a carboxyterminal nucleotide binding domain. The two subunits twist and interact with one another along the entire protein. There is considerable similarity between the structures of the nucleotide binding domains of sav1866 and other ABC transporters.
It is not fully understood how substrates are transported by sav1866. sav1866 possess a large internal cavity and a proposed transport mechanism involves two conformations of this cavity. The first conformation is inward facing, in which the cavity is open towards the interior of the cell allowing the entry of the substrate. The second conformation is outward facing, in which the cavity is open to the exterior of the cell. The binding of ATP to the nucleotide binding domain causes a tight interaction between the two domains, which induces change to outward facing conformation and permitting the exportation of substrates. The cavity returns to the inward conformation as the tight interaction between the domains is released upon the hydrolysis of ATP and its release from the nucleotide binding domain.
MATE multidrug transporters
The bacterial MATE transporters function is efflux of cationic drugs (e.g. norfloxacin and ethidium). Efflux occurs via the use of the proton motive gradient. The expression of MATE transporters occurs throughout multiple bacteria. The MATE family can be subdivided into three large subfamilies. Family 1 confers for proteins expressed that are involved in multidrug resistance (e.g. norM). The majority of MATE transporter length ranges from 400-550 amino acids, but have known to be as long as ~700 amino acids. The MATE transporters commonly contain 12 transmembrane helices. MATE transporters have been shown all to contain ~40% sequence similarity to one another; however no consensus sequence has yet been identified.
SMR transporters have shown to transport a wide range of a variety of substrates. SMR transporters confer resistance to cationic molecules, detergents, lipophilic compounds and large hydrophobic compounds. The mechanism of efflux may differ between substrates.
Not all SMR proteins have had any substrates of efflux identified, which has led to three subfamilies being classified; suppressor of groEL mutation proteins (SUG), small multidrug pumps (SMP) and paired SMR (PSMR) proteins that require the expression of two individual SMRs. Multiple SMR monomers are required for efflux of substrates. The monomers assemble to form a homo-oligomer. For efflux to occur it has been shown that at least 12 transmembranes are required, therefore SMRs must be arranged into trimers. The proposed mechanism of efflux involves the following steps: 1) exchange between substrate and a proton bound to a charged residue, 2) series of conformational changes that allow the substrate to pass through a hydrophobic channel, 3) the exchange of the substrate for a H+ in the periplasm allowing the protein to return to its original conformation.
RND superfamily efflux pumps play a vital role in multidrug resistance of gram-negative bacteria. The resistance of gram-negative bacteria is contributed to equally by the impermeability of the outer membrane and the RND transporters. RND transporters provide drug resistance by coupling to two other classes of proteins; OMF (e.g. tolC) and membrane fusion proteins (MFP) (e.g. acrA). All three components are vital for efflux of drugs to occur and if one component is absent efflux cannot take place. The formation of the tripartite complex allows drugs to be directly secreted to the external medium. The majority of transporters initially transport substrates to the periplasmic space. This direct transport means that substrates must re-penetrate the cell, which is advantageous to the bacterium.
Membrane transport proteins belonging to the MFS family are implicated in the antiport, uniport and symport of multiple compounds (e.g. sugars, Krebs cycle intermediates, phosphate esters, oligosaccharides, and antibiotics). MFS transporters can be divided into two groups consisting of 12 and 14 transmembrane segments (TMS).
Structure and Function of RND transporter acrB
An acrB monomer contains 12 transmembrane ï¡ helices. Three acrB subunits are organized into a homotrimer (Fig 1.4.). acrB subunits are comprised of a 70 Å headpiece protruding from the external membrane surface and a 50 Å thick transmembrane domain (Fig 1.4.). The top of the periplasmic domain opening is similar shape to a funnel. The periplasmic part of acrB consists of the pore domain and the tolC docking domain located farthest from the membrane plane. tolC has been shown to dock coaxially to the docking domain. Three α-helices form the pore that connects the funnel with a central cavity at the bottom of the headpiece. The central cavity leads to a further 35 Å wide transmembrane hole. The transmembrane hole has a ring like composition of helices formed by the trimer. The transmembrane hole is suggested to be occupied with phospholipids. Three chambers located above the periplasmic face lead to a central cavity. Several different hydrophobic and amphipathic ligands can bind in different positions within the acrB homotrimer cavity simultaneously. Binding involves hydrophobic forces, aromatic stacking and van der Waals interactions (Yu et al., 2003).
Figure 1.4. Visualization of multidrug efflux transporter acrB topology in bacterial gram-negative inner membrane. The three protomers of acrB which form the homotrimer are shown in green, yellow and blue at a resolution of 2.9 Å.
acrB has been shown to work in cooperation with acrA (periplasmic accessory protein) and tolC in the efflux of substrates from the cell (acrAB-tolC system). The mechanism of efflux proposed can be divided into three step rotating mechanism, whereby each monomer has one of three distinct conformations; loose (L), tight (T) and open (O). The conformation of the monomer is dependant on its functional state. Ordered binding of the substrate cause the conformational changes of each monomer (Seeger et al., (2006). The first conformation, loose, is constrained by the interactions with the two neighbouring monomers. The substrate is transferred from the transmembrane domain to the hydrophobic binding pocket in tight conformation. This conformation is induced by the neighbouring monomer being in the open state subdomains interacting. The substrate is then released towards tolC as the monomer is in the O conformation, where the pore domain is open to the exterior (Fig 1.5.) (Takatsuka and Nikaido, 2007; Takatsuka et al., 2010). The unspecific efflux mechanism proposed explains for the small molecules and wide variety of substrates exported.
Fig 1.5. Representation of the AcrB monomers in rotational transport mechanism of substrates. The conformational states loose (L), tight (T), and open (O) are shown in blue yello and red respectively.
(A). Side-view representation of two of the three monomers of the acrB trimer. acrA (green) and tolC (grey).
(B). The substrate binding sites are represented by the grooves in the L and T monomer.
The transportation of substrates is cyclical; 1) Substrate (acridine) is bound to the transmembrane domain (L conformation). 2) Substrate is transported from the transmembrane domain to the hydrophobic binding pocket converting the conformation of the monomer (T conformation). 3) Substrate is released into the funnel towards tolC (O conformation). 4) Monomer is converted from the O conformation to the initial L conformation.
Structure and function of a MFS transporter emrD
MFS transporter emrD (Multidrug resistant protein D) is a multidrug transporter from E. coli. emrD acts as an transporter for uncouplers of oxidative phosphorylation. A reduction in the proton gradient caused by oxidative phosphorylation can cause growth to arrest. emrD has been also shown to transport detergents (e.g. benzalkonium and sodium dodecylsulfate) and a range of hydrophobic compounds.
emrD has a compact structure comprised of twelve transmembrane helices spanning ~45 Å along the membrane normal and ~50 Å in the plane of the phospholipid bilayer (Fig 1.6). The internal cavity of emrD is comprised of eight helices, whilst the remaining four helices are faced outwards. The helices that form the internal cavity consist mainly of hydrophobic residues. The composition of the internal cavity is in line with the efflux of lipophillic compounds (Yin et al., 2006).
Figure 1.6. Visualization of multidrug efflux transporter emrD in bacterial gram-negative inner membrane. The red line represents the periplasmic side of the inner membrane and the blue line the cytoplasmic side.
Based on the structural homology of emrD to other MFS transporters, certain assumptions can be made about its mechanism of action. Adaptor proteins have been identified that assist the efflux of substrates through the periplasmic space for several MDR MFS systems. However, no such adaptor protein that is coupled with emrD has been identified. Alternatively emrD may act alone in a similar way to other gram-positive bacterial proteins (e.g. lmrP and bmr). emrDs intracellular loop region is comparable to the intracellular domain to that of msbA. The helices within msbA are believed to be responsible for the recognition of the head groups of substrates and for the translation of structural changes caused by ATP hydrolysis and substrate binding.
Importance of 3D Structure
For the design of newer and safer drugs that possess good potency, it is necessary to understand drug receptor interactions in detail. Drug receptor interactions are governed by the stereochemistry of the drugs and the receptors. Enhanced knowledge of drug design specifics will increase the chances of obtaining a safer drug with good therapeutic effects. Information required for this can be obtained via the resolution of the 3D structure of proteins.
The resolution of the 3D structure of proteins is a key factor in gaining more detailed understanding of the function of a protein, for example by showing ligand binding sites involved in inhibition or activation of a protein. The acquirement of the 3D structure can also show information on the mechanism of the protein.
With concerns to antibiotic resistance within bacteria a more detailed knowledge of the 3D structure can aid the development of new drugs to target these structures, as new ligand binding sites can be identified that may inhibit the action of the protein. Modified antibiotics may also be developed which are effective against bacteria with antibiotic resistant phenotypes. Patients infected with an antibiotic resistant strain of bacteria being can then be treated more efficiently.
Challenges of gaining membrane protein structure
The low abundance and the hydrophobic nature of membrane proteins conferring antibiotic resistance can make them difficult to isolate in amounts required for the elucidation of their 3D structures. The determination of inner membrane protein structures is therefore the major bottleneck in the quest to understand the detailed molecular mechanisms of membrane transport.
One of the main difficulties associated with the structural determination of membrane proteins is the solubilisation of the target protein using various detergents.dsfsf?
Protein targets of interest
Due to antibiotic resistance being a growing problem the proteins that I will be investigating are from a range of families conferring antibiotic resistance.
rarD is part of a new multidrug transporter family (eamA transporters). This family has not yet been characterised. There is a limited amount of information on the function of rarD, apart from it being responsible for chloramphenicol resistance.
tehA SMR family transporter
tehA is part of the SMR family. Resistance to most lipophillic cationic dyes, related compounds and single quaternary cation (e.g. ethidium) in E. coli is attributed to the expression of tehA. Hypersensitivity to compounds with two quaternary cations is also conferred for by the expression of tehA. This function however does not require tehB.
Localized homology of a core region (transmembrane sequences (TMS) II - V) has been shown between tehA and other SMR proteins. The core region is thought to be principally responsible for efflux conferring resistance. Deletion of the C-terminal region (TMS VIII to X) has been shown not to inhibit efflux significantly
tehB has been shown to have sequence homology to many other SAM-dependent non-nucleic acid methyltransferases. SAM methyltransferases possess three conserved motifs. tehB shares these motifs with equivalent distances between motifs. It is proposed that tehB detoxifies tellurite via its methyltransferase activity by chemically modifying tellurite with methyl groups. The final methylated tellurite product is non-volatile. The modification and removal of tellurite by tehAB occurs continuously. tehB has been found to inhibit tehA's efflux of ethidium, resulting in resistance to ethidium. It is suggested that tehB interacts with tehA and this interaction may change its affinity to certain compounds.
bcr MFS transporter
bcR belongs to the same family as emrD, the major facilitator superfamily. bcR is involved in sulfonamide and bicyclomycin resistance. bcr performs this function as it is an efflux pump for bicyclomycin, L-cysteine and sulfonamides. bcR may also be a membrane translocase (9).
baeS and components it regulates
baeS is a member of the two-component regulatory system baeS/baeR. baeS may activate baeR by phosphorylation and act as a regulator of genes that confer antibiotic resistance, including mdtABC (10). mdt B/C are part of the same superfamily as acrB (RND superfamily), whereas mdtA is part of the membrane fusion protein family (MFP). mdtA/B/C form the mdtABC tripartite complex, conferring resistance against novobiocin and deoxycholate (10).
opmN and nmpC are general outer membrane porins. Porins are beta barrel transmembrane proteins. Porins act as a pore through the membrane through which small metabolites (sugars, amino acids and ions) can diffuse, often by passive transportation.
The research to be carried out is to crystallize and obtain the 3D structures of the proteins in table 1 using x-ray crystallography. The aims of the project are: 1) express outer membrane targets, 2) purify outer membrane targets, 3) crystalise outer membrane targets, 4) increase expression of inner membrane targets to produce sufficient quantities in order for purification to obtain adequate pure protein, 5) perform FSEC and stability assays on all inner membrane targets, 6) crystalise remaining inner membrane targets, 7) reproduce highly diffracting rarD crystals, 8) characterise rarD using isothermal titration calorimetry (ITC), 9) improve tehA diffraction.
Table 1. Progress of target proteins so far.
Progress So Far
~ 4.3 Å
~ 2.6 Å
~ 6.0 Å
The outer membrane proteins will also have been included in conjunction with inner membrane proteins as they are more stable compared to inner membrane proteins, so in theory should be easier to crystallise. Outer membrane proteins can be expressed in inclusion bodies and then refolded as well as being produced in a functional form within the membrane, making them easier to crystallise.