School of Biosciences

The key cellular process of protein quality control (PQC) ensures that all proteins are structurally and functionally intact and properly localized. Although PQC is in particular demand under stress conditions, such as heat shock, it plays an important role under any environmental situation as protein misfolding occurs even under homeostatic conditions. Deficient PQC often leads to the formation of protein aggregates that are the basis for severe disorders, such as Alzheimers or Parkinsons disease. PQC is also critical for bacterial virulence, pathogenic bacteria without these cellular factors are unable to survive in their hosts. Consequently, selective inhibition of bacterial PQC might represent a promising strategy for developing novel antibiotics.

The lipoyl domains of the dihydrolipoyl acyltransferase (E2p, E2o) components of the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes are speci®cally recognised by their cognate 2-oxo acid decarboxylase (E1p, E1o). A prominent surface loop links the ®rst and second bstrands in all lipoyl domains, close in space to the lipoyl-lysine b-turn. This loop was subjected to various modi®cations by directed mutagenesis of a sub-gene encoding a lipoyl domain of Escherichia coli E2p. Deletion of the loop (four residues) rendered the domain incapable of reductive acetylation by E. coli E1p in the presence of pyruvate, but insertion of a new loop (six residues) corresponding to that in the E2o lipoyl domain partly restored this ability, albeit with a much lower rate. However, the modi®ed domain remained unable to undergo reductive succinylation by E1o in the presence of 2-oxoglutarate. Additional exchange of the two residues on the C-terminal side of the loop (V14A, E15T) had no effect. Insertion of a different four-residue loop also restored a limited ability to undergo reductive acetylation, but still signi®cantly less than that of the wild-type domain. Exchanging the residue on the N-terminal side of the lipoyl-lysine b-turn in the E2p and E2o domains (G39T), both singly and in conjunction with the loop exchange, had no effect on the ability of the E2p domain to be reductively acetylated but did confer a slight increase in susceptibility to reductive succinylation. All mutant E2p domains, apart from that with the loop deletion (LD), were readily lipoylated in vitro by E. coli lipoate protein ligase A; the E2p LD mutant could be lipoylated only at a signi®cantly lower rate. Likewise, this domain exhibited 1D and 2D NMR spectra characteristic of a partially folded protein, whereas the spectra of mutants with modi®ed loops were similar to those of the wild-type domain. The surface loop is evidently important to the structural integrity of the domain and may help to stabilize the thioester bond linking the acyl group to the reduced lipoyl-lysine swinging arm as part of the catalytic mechanism. Recognition of the lipoyl domain by its partner E1 appears to be a complex process and not attributable to any single determinant on the domain.

Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeqNNN. The bla gene encoding TEM-1 β-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.

The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 β-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli. Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10000 μg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of α-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.

Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed speci®cally by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex. Residues in the lipoyl-lysine b-turn region of the unlipoylated E2plip domain (E2plip apo ) undergo signi®cant changes in both chemical shift and transverse relaxation time (T 2 ) in the presence of E1p but not E1o. Residue Gly11, in a prominent surface loop between b-strands 1 and 2 in the E2plip domain, was also observed to undergo a signi®cant change in chemical shift. Addition of pyruvate to the mixture of E2plip apo and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both b-strands 4 and 5, together with those in the prominent surface loop and the following b-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31. The values of T 2 across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip apo binds more tightly after the addition of pyruvate. The lipoylated domain (E2plip holo ) also exhibited signi®cant changes in chemical shift and decreases in the overall T 2 relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain that contains the lipoyl-lysine (Lys41) residue. In addition, small chemical shift changes and a general drop in T 2 times in the presence of E1o were observed, indicating that E2plip holo can interact, weakly but non-productively, with E1o. It is evident that recognition of the protein domain is the ultimate determinant of whether reductive acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the Elp.

While the deletion of an amino acid is a common mutation observed in nature, it is generally thought to be disruptive to protein structure. Using a directed evolution approach, we find that the enzyme TEM-1 b-lactamase was broadly tolerant to the deletion mutations sampled. Circa 73% of the variants analysed retained activity towards ampicillin, with deletion mutations observed in helices and strands as well as regions important for structure and function. Several deletion variants had enhanced activity towards ceftazidime compared to the wild-type TEM-1 demonstrating that removal of an amino acid can have a beneficial outcome.

Cytochrome b 562 was engineered to introduce a cysteine residue at a surface-exposed position to facilitate direct selfassembly on a Au(111) surface. The confined protein exhibited reversible and fast electron exchange with a gold substrate over a distance of 20 Å between the heme redox center and the gold surface, a clear indication that a long-range electron-transfer pathway is established. Electrochemical scanning tunneling microscopy was used to map electron transport features of the protein at the single-molecule level. Tunneling resonance was directly imaged and apparent molecular conductance was measured, which both show strong redox-gated effects. This study has addressed the first case of heme proteins and offered new perspectives in singlemolecule bioelectronics.

A directed evolution method has been developed that allows random substitution of a contiguous trinucleotide sequence for TAG throughout a target gene for use in conjunction with an expanded genetic code. Using TEM-1 b-lactamase and enhanced green fluorescent protein as targets, protein variants were identified whose functional phenotype was rescued in vivo when co-expressed with orthogonal tRNA-aminoacyl-tRNA synthase pairs that insert p-iodophenylalanine in response to UAG. Sequencing of the selected clones that retained the target protein function revealed that 490% of the variants contained in-frame TAG codons distributed throughout the target gene. Such an approach will allow broader sampling of new chemical diversity by proteins, so opening new avenues for studying biological systems and for adapting proteins for biotechnological applications. A common set of reagents allows the method to be used on different protein systems and in combination with an array of different unnatural amino acids, so helping to reveal the true potential for engineering proteins through expanded chemical diversity sampling.

Combinatorial fragment exchange was utilised to recombine key structural and functional low homology regions of bacilli subtilisins to generate new active hybrid proteases with altered substrate profiles. Up to six different regions comprising mostly of loop residues from the commercially important subtilisin Savinase were exchanged with the structurally equivalent regions of six other subtilisins. The six additional subtilisins derive from diverse origins and included thermophilic and intracellular subtilisins as well as other academically and commercially relevant subtilisins. Savinase was largely tolerant to fragment exchange; rational replacement of all six regions with 5 of 6 donating subtilisin sequences preserved activity, albeit reduced compared to Savinase. A combinatorial approach was used to generate hybrid Savinase variants in which the sequences derived from all seven subtilisins at each region were recombined to generate new region combinations. Variants with different substrate profiles and with greater apparent activity compared to Savinase and the rational fragment exchange variants were generated with the substrate profile exhibited by variants dependent on the sequence combination at each region.

The plant vigour hypothesis proposes that herbivores should favour feeding on more vigorously growing plants or plant modules. Similarly, we would expect herbivores to favour plants that regrow vigorously after herbivory. Larger animals, like elephants, may also select plant species relative to their availability and prefer species with larger growth forms in order to meet their intake requirements. The food preferences of the Bornean elephant (Elephas maximus borneensis) in the Lower Kinabatangan Wildlife Sanctuary, Sabah, Malaysia, were investigated along 12 transects in areas where elephants were recently sighted feeding. One hundred and eighty-two plants were eaten and 185 plants were measured for species availability along transects. Species vigour was determined by the monthly regrowth in new shoot length after elephant feeding and the number of new shoots produced on each plant. Measurements were carried out on each plant for 9 mo or until the new shoot was eaten. Plant sizes were determined from their basal diameter. The Bornean elephant did not prefer more vigorous species or species with larger growth forms. New shoots did not grow longer on preferred than avoided species. Additionally, unlike other elephants that live in a forest environment, the Bornean elephant preferred species from the Poaceae (specifically Phragmites karka and Dinochloa scabrida) over other plant types including gingers, palms, lianas and woody trees.

This paper reports in detail, for the first time, on two anesthetic procedures performed on a 15-yr-old, 530 kg, adult male Sumatran rhinoceros (Dicerorhinus sumatrensis). The anesthetic procedures were carried out in order to perform semen collection via electro-ejaculation, using well-established methods, and in order to examine and sample bilateral corneal opacities. Anesthesia for the first procedure was induced with a combination of 5 mg (0.0094 mg/kg) butorphanol tartrate and 5 mg (0.0094 mg/kg) detomidine hydrochloride administered intramuscularly. Subsequently, 0.74 mg (0.0014 mg/kg) etorphine and 3 mg (0.0057 mg/kg) acepromazine, with an additional 1.5 mg butorphanol (0.0028 mg/kg) and 1.5 mg (0.0028 mg/kg) detomidine, were administered intramuscularly. The second procedure was carried out using an intramuscular combination of butorphanol (0.019 mg/kg) and detomidine (0.019 mg/kg), followed by etorphine (0.0023 mg/kg) and acepromazine (0.009 mg/ kg). During the second procedure, the depth of anesthesia was managed with very small, supplemental intravenous doses of 50 mg ketamine (0.094 mg/kg). Sequential arterial blood gas analysis demonstrated respiratory acidosis with hypoxemia. Heart rate and respiratory rate ranged between 60-74 beats per minute (bpm), and 10-20 breaths per minute, respectively. Reversal after 100 min, with the intravenous administration of 150 mg (0.28 mg/kg) naltrexone and intravenous 20 mg (0.038 mg/kg) atipamezole, was uneventful and rapid, with the animal standing after 2 min. This combination provides satisfactory general anesthesia in this critically endangered species and will facilitate veterinary management of this species in captivity.

Recursion by herbivores is the repeated use of the same site or plants. Recursion by wild animals is rarely investigated but may be ubiquitous. Optimal foraging theory predicts site recursion as a function of the quality of the site, extent of its last use, and time since its last use because these influence site resource status and recovery. We used GPS collars, behaviour and site sampling to investigate recursion to foraging sites for two elephant Elephas maximus borneensis herds in the Lower Kinabatangan Wildlife Sanctuary, Borneo, over a 12 month period. Recursion occurred to 48 out of 87 foraging sites and was most common within 48 hours or between 151–250 days, indicating two different types of recursion. Recursion was more likely to occur if the site had previously been occupied for longer. Moreover, the time spent at a site at recursion was the same as the time spent at the site on the first occasion. The number of days that had passed between the first visit and recursion w...

Understanding the natural diet of species may provide useful information that can contribute to successful captive maintenance. A common problem experienced with captive foregut-fermenting primate (colobine) diets is that they are deficient in fiber and therefore highly digestible. This may contribute to gastrointestinal disorders often observed in zoos. An approach to obtain information relevant for the improvement of diets is to compare the nutrient composition of feces from free-ranging and captive individuals. In theory, fecal material can be considered a proxy for diet intake integrated over a certain period of time. We collected fecal samples from eight free-ranging proboscis monkey (Nasalis larvatus, a highly endangered colobine species) groups from a secondary forest along the Kinabatangan River and four from a mixed mangrove-riverine forest along the Garama River, Sabah, Borneo, Malaysia. We also collected fecal samples from 12 individual captive adult/sub-adult proboscis monkeys from three different zoos. We confirmed that feces from free-ranging monkeys contained more fiber and less metabolic fecal nitrogen than those from captive specimens, indicating a less digestible diet in the wild. Modifying the diets of captive colobines to include more fiber, comparable to those of free-ranging ones, may contribute to their health and survival.

The Lkb1 tumour suppressor is a multitasking kinase participating in a range of physiological processes. We have determined the impact of Lkb1 deficiency on intestinal homeostasis, particularly focussing on secretory cell differentiation and development since we observe strong expression of Lkb1 in normal small intestine Paneth and goblet cells. We crossed mice bearing an Lkb1 allele flanked with LoxP sites with those carrying a Cyp1a1-specific inducible Cre recombinase. Lkb1 was efficiently deleted from the epithelial cells of the mouse intestine after intraperitoneal injection of the inducing agent bnaphthoflavone. Bi-allelic loss of Lkb1 led to the perturbed development of Paneth and goblet cell lineages. These changes were characterised by the lack of Delta ligand expression in Lkb1-deficient secretory cells and a significant increase in the levels of the downstream Notch signalling effector Hes5 but not Hes1. Our data show that Lkb1 is required for the normal differentiation of secretory cell lineages within the intestine, and that Lkb1 deficiency modulates Notch signalling modulation in post-mitotic cells.

CLIC proteins are components or regulators of novel intracellular anion channels in mammalian cells, and previous studies have suggested that human nuclear membrane-associated CLIC1 and mouse inner mitochondrial membrane CLIC4 are involved in cell division and apoptosis. We have isolated Xenopus homologues of CLIC1 and CLIC4 and shown them to be well conserved during chordate evolution, but poorly conserved in invertebrates. Consistent with fundamental cellular roles, Xenopus CLIC genes are expressed at every stage of embryonic development. Expression is localised to mesodermal and ectodermal tissues, with particularly marked expression of xCLIC4 in the developing nervous system. This is the first description of non-mammalian CLIC expression, and use of Xenopus laevis as a model organism may provide insights into the role of CLIC-associated ion channels in animal development.

The AKT/PI3K/mTOR pathway is frequently altered in a range of human tumours, including bladder cancer. Here we report the phenotype of mice characterised by deletion of two key players in mTOR regulation, Pten and Lkb1, in a range of tissues including the mouse urothelium. Despite widespread recombination within the range of epithelial tissues, the primary phenotype we observe is the rapid onset of bladder tumorigenesis, with median onset of approximately 100 days. Single deletion of either Pten or Lkb1 had no effect on bladder cell proliferation or tumour formation. However, simultaneous deletion of Lkb1 and Pten led to an upregulation of the mTOR pathway and the hypoxia marker GLUT1, increased bladder epithelial cell proliferation and ultimately tumorigenesis. Bladder tissue also exhibited characteristic features of epithelialmesenchymal transition, with loss of the epithelial markers E-cadherin and the tight junction protein ZO-1, and increases in the mesenchymal marker vimentin as well as nuclear localization of epithelial-mesenchymal transition (EMT) regulator Snail. We show that these effects were all dependent upon mTOR activity, as rapamycin treatment blocked both EMT and tumorigenesis. Our data therefore establish clear synergy between Lkb1 and Pten in controlling the mTOR pathway within bladder epithelium, and show that loss of this control leads to the disturbance of epithelial structure, EMT and ultimately tumorigenesis.