Dataset Information

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.

ABSTRACT: P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysis-driven drug export in Pgp are poorly understood. Fluorescence spectroscopy using tryptophans (Trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important for function, protein-lipid interactions, and/or protein stability. Here, we developed a directed evolutionary approach to first replace all eight transmembrane Trps and select for transport-active mutants in Saccharomyces cerevisiae. Surprisingly, many Trp positions contained non-conservative substitutions that supported in vivo activity, and were preferred over aromatic amino acids. The most active construct, W(3Cyto), served for directed evolution of the three cytoplasmic Trps, where two positions revealed strong functional bias towards tyrosine. W(3Cyto) and Trp-less Pgp retained wild-type-like protein expression, localization and transport function, and purified proteins retained drug stimulation of ATP hydrolysis and drug binding affinities. The data indicate preferred Trp substitutions specific to the local context, often dictated by protein structural requirements and/or membrane lipid interactions, and these new insights will offer guidance for membrane protein engineering.

SUBMITTER: Swartz DJ

PROVIDER: S-EPMC7035247 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.

Swartz Douglas J DJ Singh Anukriti A Sok Narong N Thomas Joshua N JN Weber Joachim J Urbatsch Ina L IL

Scientific reports 20200221 1

P-glycoprotein (Pgp) pumps an array of hydrophobic compounds out of cells, and has major roles in drug pharmacokinetics and cancer multidrug resistance. Yet, polyspecific drug binding and ATP hydrolysis-driven drug export in Pgp are poorly understood. Fluorescence spectroscopy using tryptophans (Trp) inserted at strategic positions is an important tool to study ligand binding. In Pgp, this method will require removal of 11 endogenous Trps, including highly conserved Trps that may be important fo ...[more]

PMID: 32081894

Similar Datasets

Project description:HIV-1 envelope glycoprotein (Env) glycosylation is important because individual glycans are components of multiple broadly neutralizing antibody epitopes, while shielding other sites that might otherwise be immunogenic. The glycosylation on Env is influenced by a variety of factors, including the genotype of the protein, the cell line used for its expression, and the details of the construct design. Here, we used a mass spectrometry (MS)-based approach to map the complete glycosylation profile at every site in multiple HIV-1 Env trimers, accomplishing two goals. (i) We determined which glycosylation sites contain conserved glycan profiles across many trimeric Envs. (ii) We identified the variables that impact Env's glycosylation profile at sites with divergent glycosylation. Over half of the gp120 glycosylation sites on 11 different trimeric Envs have a conserved glycan profile, indicating that a native consensus glycosylation profile does indeed exist among trimers. We showed that some soluble gp120s and gp140s exhibit highly divergent glycosylation profiles compared to trimeric Env. We also assessed the impact of several variables on Env glycosylation: truncating the full-length Env; producing Env, instead of the more virologically relevant T lymphocytes, in CHO cells; and purifying Env with different chromatographic platforms, including nickel-nitrilotriacetic acid (Ni-NTA), 2G12, and PGT151 affinity. This report provides the first consensus glycosylation profile of Env trimers, which should serve as a useful benchmark for HIV-1 vaccine developers. This report also defines the sites where glycosylation may be impacted when Env trimers are truncated or produced in CHO cells.IMPORTANCE A protective HIV-1 vaccine will likely include a recombinant version of the viral envelope glycoprotein (Env). Env is highly glycosylated, and yet vaccine developers have lacked guidance on how to assess whether their immunogens have optimal glycosylation. The following important questions are still unanswered. (i) What is the "target" glycosylation profile, when the goal is to generate a natively glycosylated protein? (ii) What variables exert the greatest influence on Env glycosylation? We identified numerous sites on Env where the glycosylation profile does not deviate in 11 different Env trimers, and we investigated the impact on the divergent glycosylation profiles of changing the genotype of the Env sequence, the construct design, the purification method, and the producer cell type. The data presented here give vaccine developers a "glycosylation target" for their immunogens, and they show how protein production variables can impact Env glycosylation.

Project description:ObjectiveThe aim of the current study was to evaluate the effects of and adherence to an active video game promotion intervention on anthropometrics, sedentary screen time and consumption of sugar-sweetened beverages and snacks among non-active video gaming adolescents who primarily were of healthy weight.MethodsWe assigned 270 gaming (i.e. ? 2 hours/week non-active video game time) adolescents randomly to an intervention group (n = 140) (receiving active video games and encouragement to play) or a waiting-list control group (n = 130). BMI-SDS (SDS = adjusted for mean standard deviation score), waist circumference-SDS, hip circumference and sum of skinfolds were measured at baseline, at four and ten months follow-up (primary outcomes). Sedentary screen time, physical activity, consumption of sugar-sweetened beverages and snacks, and process measures (not at baseline) were assessed with self-reports at baseline, one, four and ten months follow-up. Multi-level-intention to treat-regression analyses were conducted.ResultsThe control group decreased significantly more than the intervention group on BMI-SDS (? = 0.074, 95%CI: 0.008;0.14), and sum of skinfolds (? = 3.22, 95%CI: 0.27;6.17) (overall effects). The intervention group had a significantly higher decrease in self-reported non-active video game time (? = -1.76, 95%CI: -3.20;-0.32) and total sedentary screen time (Exp (? = 0.81, 95%CI: 0.74;0.88) than the control group (overall effects). The process evaluation showed that 14% of the adolescents played the Move video games every week ? 1 hour/week during the whole intervention period.ConclusionsThe active video game intervention did not result in lower values on anthropometrics in a group of 'excessive' non-active video gamers (mean ~ 14 hours/week) who primarily were of healthy weight compared to a control group throughout a ten-month-period. Even some effects in the unexpected direction were found, with the control group showing lower BMI-SDS and skin folds than the intervention group. The intervention did result in less self-reported sedentary screen time, although these results are likely biased by social desirability.Trial registrationDutch Trial Register NTR3228.

Dataset Information

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.

Publications

Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets