Protein and Drug Design Algorithms Using Improved Biophysical Modeling

This thesis focuses on the development of algorithms that will allow protein design calculations to incorporate more realistic modeling assumptions. Protein design algorithms search large sequence spaces for protein sequences that are biologically and medically useful. Better modeling could improve the chance of success in designs and expand the range of problems to which these algorithms are applied. I have developed algorithms to improve modeling of backbone flexibility (DEEPer) and of more extensive continuous flexibility in general (EPIC and LUTE). I’ve also developed algorithms to perform multistate designs, which account for effects like specificity, with provable guarantees of accuracy (COMETS), and to accommodate a wider range of energy functions in design (EPIC and LUTE).

Studies of Protein-Protein and Protein-Water Interactions by Small Angle X-Ray Scattering, Terahertz Spectroscopy, ASMOS, And Computer Simulation

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 µm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.

Responses to various protein and energy supplements by steers fed low-quality tropical hay. 1. Comparison of response surfaces for young steers

Response curves were established for different supplements, offered at intakes ranging from 0 to 20 g/kg liveweight (W).day to young Bos indicus crossbred steers fed low-quality Rhodes grass (Chloris gayana) hay ad libitum in two pen experiments. Supplements included protein meals of varying rumen-degradability (cottonseed meal (CSM) or fishmeal), as well as ‘energy sources’ comprising grains of high and low ruminal starch degradability (barley and sorghum) and a highly fermentable sugar source (molasses), with all diets adjusted for rumen-degradable nitrogen and mineral content. Unsupplemented steers gained 0.08 and 0.15 kg/day, in Experiments 1 and 2, respectively. Growth of steers increased linearly with intake of ‘energy source’ supplements in increasing order of molasses, sorghum and barley (all differences P < 0.05). Steer growth rate also increased linearly with fishmeal, albeit over a narrow intake range (0–4.1 g/kg W.day), whereas the response with CSM was asymptotic, showing a steep response at low intake before levelling at ~1.2 kg/day. All supplement types were associated with a linear reduction in hay intake by the steers (energy substitution) where the reduction was greater (P < 0.05) for barley and molasses (not different) than for sorghum (P < 0.05), and for fishmeal compared with CSM (P < 0.05). In concurrent metabolism studies with the same rations, organic matter digestibility of the total ration (561–578 g/kg DM, unsupplemented) was increased linearly by barley and molasses (both P < 0.05) but was unaffected by CSM and sorghum supplements. The efficiency of microbial protein synthesis in steers increased linearly, from 91 g microbial crude protein/kg digestible organic matter (unsupplemented), in both molasses and CSM-supplemented steers, with the trend for a higher response to molasses (P = 0.05), and appeared most closely related to digestible organic matter intake. The response curves from these studies provide the practical framework upon which to formulate rations for cattle grazing low-quality forages.

Responses to various protein and energy supplements by steers fed low-quality tropical hay. 2. Effect of stage of maturity of steers

Dose response curves to various supplements were established in two pen-feeding experiments (Exp1 and Exp2) with Bos indicus crossbred steers of two age groups (Young, 10–12 months; Old, 33–36 months) fed low-quality tropical grass hays ad libitum. Diets included supplements based on (Exp1) cottonseed meal (CSM; intake (as fed) 0–10 g/kg liveweight (W).day) and a barley mix (Bar; 0–20 g/kg W.day) and (Exp2) a molasses mix (MUP) and a Bar mix, both fed at 0–20 g/kg W.day. Urea was provided with the Bar mixes and urea/copra meal with the MUP mix. Growth rates of Young steers increased linearly with Bar and MUP supplements but asymptotically with CSM whereas those of Old steers increased asymptotically with all supplement types. With supplement intake expressed on a liveweight basis (g/kg W.day), responses were greater for both steer age groups with CSM compared with Bar (Young, P < 0.001; Old, P < 0.01) and Bar compared with MUP treatments (Young, P < 0.01; Old, P < 0.05). Furthermore, Old steers outperformed their Young counterparts with both CSM (P < 0.05) and Bar (P < 0.001) supplements fed in Exp1 and with Bar and MUP supplements (P < 0.01) fed in Exp2. When supplement intake was expressed in absolute terms (kg/day), growth responses were not different between age groups for different supplements except that Old steers had a higher daily W gain on Bar than their Young counterparts (P < 0.05). Intake of hay (W-corrected) was higher for Young compared with Old steers without supplement but was variably reduced for both steer groups with increasing supplement intake. The results of these experiments have implications for supplement formulation for steers at different stages of maturity grazing low-quality forages.

Multi-­‐dimensional Protein Identification Technology

Before the rise of the Multidimentional Protein Identification Technology (MudPIT), protein and peptide mixtures were resolved using traditional proteomic technologies like the gel-­‐ based 2D chromatography that separates proteins by isoelectric point and molecular weight. This technique was tedious and limited, since the characterization of single proteins required isolation of protein gel spots, their subsequent proteolyzation and analysis using Matrix-­‐ assisted laser desorption/ionization-­‐time of flight (MALDI-­‐TOF) mass spectrometry.

A Staggered Decameric Assembly of Human C-Reactive Protein Stabilized by Zinc Ions Revealed by X-ray Crystallography

International audience

A genetic variant of the CAPN10 gene in Mexican subjects with dyslipidemia is associated with increased HDL-cholesterol concentrations after the consumption of a soy protein and soluble fiber dietary portfolio

Dyslipidemia is a major public health problem, and therefore, it is important to develop dietary strategies to diminish the prevalence of this disorder. It was recently reported that diet may play an important role in triggering insulin resistance by interacting with genetic variants at the CAPN10 gene locus in patients with metabolic syndrome. Nonetheless, it remains unknown whether genetic variants of genes involved in the development of type 2 diabetes are associated with variations in high-density lipoprotein cholesterol (HDL-C). The study used a single-center, prospective, cohort design. Here, we assessed the effect of four variants of the CAPN10 gene on HDL-C levels in response to a soy protein and soluble fiber dietary portfolio in subjects with dyslipidemia. In 31 Mexican dyslipidemic individuals, we analyzed four CAPN10 gene variants (rs5030952, rs2975762, rs3792267, and rs2975760) associated with type 2 diabetes. Subjects with the GG genotype of the rs2975762 variant of the CAPN10 gene were better responders to dietary intervention, showing increased HDL-C concentrations from the first month of treatment. HDL-C concentrations in participants with the wild type genotype increased by 17.0%, whereas the HDL-C concentration in subjects with the variant genotypes increased by only 3.22% (p = 0.03); the low-density lipoprotein cholesterol levels of GG carriers tended to decrease (-12.6%). These results indicate that Mexican dyslipidemic carriers of the rs2975762-GG genotype are better responders to this dietary intervention.

Biochemical studies of postsynaptic density signaling proteins with a focus on synaptic GTPase activating protein and PDZ domains

Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP’s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP’s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.

The postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.

Apparent digestibility of dry matter, crude protein, and amino acids of six rendered by-products in juvenile Litopenaeus vannamei

Apparent digestibility coefficients (ADC) of dry matter, crude protein (CP), and amino acids (AA) were evaluated in diets with six rendered by-products used to feed juvenile Pacific white shrimp: two poultry meals (poultry meal 1, 69% CP; poultry meal 2, 72% CP), two feather meals (89% CP), one blood meal (96% CP), and one pork meal (57% CP). Experimental diets were formulated with 30% of the test ingredient and 70% of a commercial diet supplemented with 1% of chromium oxide as inert marker. AA contents in ingredients, diets, leached diets, and feces were determined by high performance liquid chromatography. Preprandial AA losses attributed to leaching were higher in the blood meal diet (15%) and pork meal diet (10%). Poultry meal diets 1 and 2 showed mean AA losses of 3% and 5%, respectively, while the reference diet had a mean AA leaching of 6%. The AA that had the highest leaching rates were lysine (21%), methionine (15%), and histidine (12%). The ADC of dry matter was higher for poultry meals 1 (70%) and 2 (73%), followed by pork meal (69%), feather meals (61%), and blood meal (57%). The digestibility of CP was higher for poultry meals (78–80%), followed by pork meal (76%), and blood meal and feather meals (65–67%). The digestibility of CP in the reference diet (83%) was higher than that observed for all the animal by-product meals except the poultry meals. The ADC of the sum of AA adjusted for nutrient leaching fluctuated from 65% for blood meal to 80% for poultry meals.

Growth, lipogenesis and body composition of piracanjuba (Brycon orbignyanus) fingerlings fed different dietary protein and lipid concentrations

Piracanjuba (Brycon orbignyanus) is a Brazilian migratory fast-growing omnivore, very appreciated as a sport fish, which is threatened to extinction in Southern Brazil due to stock over exploitation and dam building. Therefore, efforts have been made to raise this fish in captivity for reintroduction and aquaculture purposes. In the present study, the effects of different dietary protein and lipid concentrations on piracanjuba fingerlings growth performance, feed utilization, body composition, hepatosomatic index (HSI) and activity of the lipogenic enzymes fatty acid synthetase (FAS), glucose-6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) were investigated using a 2 x 3 factorial experiment. Six casein-gelatin based diets were prepared combining two protein (30% and 32%) and three lipid concentrations (5.5%, 8.8% and 12.1%). Eleven fish, average weight 11.30 +/- 0.1 g, were held in each of 18 100-1 aquaria, supplied with recirculating freshwater. Each diet was randomly assigned to triplicate groups of fish and fed to apparent satiation, twice a day for 100 d. Piracanjuba fingerlings' daily weight gain (0.36-0.40 g), specific growth rate (1.43-1.51%), feed utilization and HSI were not influenced by dietary protein or lipid concentration. However, body composition was directly affected by dietary treatment. An increase in body fat and dry matter was observed as dietary lipid increased, for both dietary protein concentrations tested. The activity of FAS was depressed by increasing dietary fat levels but the G6PD activity did not differ among dietary treatments, although ME activity showed some regulation by dietary protein. These results indicate that an increase from 5.5% to 12.1% in the dietary lipid, at a dietary protein concentration of 30% or 32%, promotes body fat accumulation in piracanjuba fingerlings with no improvement in growth, suggesting that the lipid requirement for this species should be 5% or less, when raised for commercial purposes. However, the additional energy reserve from body fat accumulation could be desirable for piracanjuba fingerlings produced for stock enhancement. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.

Microfluidics based manufacture of liposomes simultaneously entrapping hydrophilic and lipophilic drugs

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300 nm. In terms of drug loading, microfluidics production promoted high loading within ∼100 nm vesicles for both the water soluble drug (20–25% of initial amount added) and the bilayer embedded drug (40–42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug.

Snake Venom L-Amino Acid Oxidases: Some Consideration About their Functional Characterization

Snake Venom L-amino acid oxidases (LAAOs E.C. 1.4.3.2) are flavoenzymes broadly found in various snake venom compositions. LAAOs have become an attractive subject for molecular biology, biochemistry, physiology and medicine due to their actions on various cells and biological effects on platelets, apoptosis, hemorrhage and others. In this review we try to summarize some of these reports, with special emphasis on apoptosis, anti-protozoa, bactericidal and anti-viral activities.

Venom-Sweet-Venom: N-Linked Glycosylation in Snake Venom Toxins

Protein glycosylation represents one of the most important post-translational events, and is a mean of diversifying a protein without recourse to the genome. The venoms produced by snakes contain an abundance of glycoproteins with N-linked carbohydrates. N-linked glycosylation can ensure the correct folding of important functional domains. Characterization of carbohydrates structures aids in development of human therapeutics by snake venom toxins.