A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3′ - 5′)-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable ‘module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protei

Novel olfactory ligands via terpene synthases

A synthetic biology approach to the rational design of analogues of olfactory ligands by providing unnatural substrates for the enzyme synthesising (S)-germacrene D, an olfactory ligand acting as a plant derived insect repellent, to produce novel ligands is described as a viable alternative to largely unsuccessful ligand docking studies. (S)-14,15-Dimethylgermacrene D shows an unexpected reversal in behavioural activity

Modular chemoenzymatic synthesis of terpenes and their analogues

Non‐natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large‐scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)‐FDP and (2Z,6Z)‐FDP, were isolated in greater than 80 % yields. The synthesis of 14,15‐dimethyl‐FDP, 12‐methyl‐FDP, 12‐hydroxy‐FDP, homo‐FDP, and 15‐methyl‐FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)‐14,15‐dimethylgermacrene D and (S)‐12‐methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non‐natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions

Loop Interactions during Catalysis by Dihydrofolate Reductase fromMoritella profunda

Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the wellstudied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR

Feasibility of joystick guided colonoscopy

The flexible endoscope is increasingly used to perform minimal invasive interventions. A novel add-on platform allows single-person control of both endoscope and instrument at the site of intervention. The setup changes the current routine of handling the endoscope. This study aims to determine if the platform allows effective and efficient manipulation to position the endoscope at potential intervention sites throughout the bowel. Five experts in flexible endoscopy first performed three colonoscopies on a computer simulator using the conventional angulation wheels. Next they trained with the joystick interface to achieve their personal level of intubation time with low pain score. 14 PhD students (novices) without hands-on experience performed the same colonoscopy case using either the conventional angulation wheels or joystick interface. Both novice groups trained to gain the average expert level. The cecal intubation time, pain score and visualization performance (% of bowel wall) were recorded. All experts reached their personal intubation time in 6 ± 6 sessions. Three experts completed their learning curve with low pain score in 8 ± 6 sessions. The novices required 11 ± 6 sessions using conventional angulation wheels, and 12 ± 6 sessions using the joystick interface. There was no difference in the visualization performance between the novice and between the expert groups. This study shows that the add-on platform enables endoscope manipulation required to perform colonoscopy. Experts need only a relatively short training period. Novices are as effective and as efficient in endoscope manipulation when comparing the add-on platform with conventional endoscope contro

Linking the functions of unrelated proteins using a novel directed evolution domain insertion method

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

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy (15N, 13C, 2H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for “promoting motions” in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate

Thermal Adaptation of Dihydrofolate Reductase from the Moderate ThermophileGeobacillus stearothermophilus

The thermal melting temperature of dihydrofolate reductase from Geobacillus stearothermophilus (BsDHFR) is 30 °C higher than that of its homologue from the psychrophile Moritella profunda. Additional proline residues in the loop regions of BsDHFR have been proposed to enhance the thermostability of BsDHFR, but site-directed mutagenesis studies reveal that these proline residues contribute only minimally. Instead, the high thermal stability of BsDHFR is partly due to removal of water-accessible thermolabile residues such as glutamine and methionine, which are prone to hydrolysis or oxidation at high temperatures. The extra thermostability of BsDHFR can be obtained by ligand binding, or in the presence of salts or cosolvents such as glycerol and sucrose. The sum of all these incremental factors allows BsDHFR to function efficiently in the natural habitat of G. stearothermophilus, which is characterized by temperatures that can reach 75 °C

Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei

Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target