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Cytosolic selection systems to study protein stability.

ABSTRACT: Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge.

SUBMITTER: Malik A

PROVIDER: S-EPMC4248860 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Cytosolic selection systems to study protein stability.

Malik Ajamaluddin A Mueller-Schickert Antje A Bardwell James C A JC

Journal of bacteriology 20140929 24

Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within gue ...[more]

PMID: 25266385

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Project description:Despite intense work, incorporating constraints on protein native structures into the mathematical models of molecular evolution remains difficult, because most models and programs assume that protein sites evolve independently, whereas protein stability is maintained by interactions between sites. Here, we address this problem by developing a new mean-field substitution model that generates independent site-specific amino acid distributions with constraints on the stability of the native state against both unfolding and misfolding. The model depends on a background distribution of amino acids and one selection parameter that we fix maximizing the likelihood of the observed protein sequence. The analytic solution of the model shows that the main determinant of the site-specific distributions is the number of native contacts of the site and that the most variable sites are those with an intermediate number of native contacts. The mean-field models obtained, taking into account misfolded conformations, yield larger likelihood than models that only consider the native state, because their average hydrophobicity is more realistic, and they produce on the average stable sequences for most proteins. We evaluated the mean-field model with respect to empirical substitution models on 12 test data sets of different protein families. In all cases, the observed site-specific sequence profiles presented smaller Kullback-Leibler divergence from the mean-field distributions than from the empirical substitution model. Next, we obtained substitution rates combining the mean-field frequencies with an empirical substitution model. The resulting mean-field substitution model assigns larger likelihood than the empirical model to all studied families when we consider sequences with identity larger than 0.35, plausibly a condition that enforces conservation of the native structure across the family. We found that the mean-field model performs better than other structurally constrained models with similar or higher complexity. With respect to the much more complex model recently developed by Bordner and Mittelmann, which takes into account pairwise terms in the amino acid distributions and also optimizes the exchangeability matrix, our model performed worse for data with small sequence divergence but better for data with larger sequence divergence. The mean-field model has been implemented into the computer program Prot_Evol that is freely available at http://ub.cbm.uam.es/software/Prot_Evol.php.

Project description:Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

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Cytosolic selection systems to study protein stability.

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Cytosolic selection systems to study protein stability.

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