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Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.

ABSTRACT: Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric under physiologic conditions. Membrane-associated FP-fusion proteins, by virtue of their constrained planar geometry, achieve high effective concentrations. We exploited this propensity to develop an assay to measure FP tendencies to oligomerize in cells. FPs were fused on the cytoplasmic end of an endoplasmic reticulum (ER) signal-anchor membrane protein (CytERM) and expressed in cells. Cells were scored based on the ability of CytERM to homo-oligomerize with proteins on apposing membranes and restructure the ER from a tubular network into organized smooth ER (OSER) whorl structures. The ratio of nuclear envelope and OSER structures mean fluorescent intensities for cells expressing enhanced green fluorescent protein (EGFP) or monomeric green fluorescent protein (mGFP) CytERM established standards for comparison of uncharacterized FPs. We tested three FPs and identified two as sufficiently monomeric, while a third previously reported as monomeric was found to strongly oligomerize.

SUBMITTER: Costantini LM

PROVIDER: S-EPMC3324619 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.

Costantini Lindsey M LM Fossati Matteo M Francolini Maura M Snapp Erik Lee EL

Traffic (Copenhagen, Denmark) 20120220 5

Several fluorescent proteins (FPs) are prone to forming low-affinity oligomers. This undesirable tendency is exacerbated when FPs are confined to membranes or when fused to naturally oligomeric proteins. Oligomerization of FPs limits their suitability for creating fusions with proteins of interest. Unfortunately, no standardized method evaluates the biologically relevant oligomeric state of FPs. Here, we describe a quantitative visual assay for assessing whether FPs are sufficiently monomeric un ...[more]

PMID: 22289035

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Project description:Salinity majorly hinders horizontal and vertical expansion in worldwide wheat production. Productivity can be enhanced using salt-tolerant wheat genotypes. However, the assessment of salt tolerance potential in bread wheat doubled haploid lines (DHL) through agro-physiological traits and stress-related gene expression analysis could potentially minimize the cost of breeding programs and be a powerful way for the selection of the most salt-tolerant genotype. We used an extensive set of agro-physiologic parameters and salt-stress-related gene expressions. Multivariate analysis was used to detect phenotypic and genetic variations of wheat genotypes more closely under salinity stress, and we analyzed how these strategies effectively balance each other. Four doubled haploid lines (DHLs) and the check cultivar (Sakha93) were evaluated in two salinity levels (without and 150 mM NaCl) until harvest. The five genotypes showed reduced growth under 150 mM NaCl; however, the check cultivar (Sakha93) died at the beginning of the flowering stage. Salt stress induced reduction traits, except the canopy temperature and initial electrical conductivity, which was found in each of the five genotypes, with the greatest decline occurring in the check cultivar (Sakha-93) and the least decline in DHL2. The genotypes DHL21 and DHL5 exhibited increased expression rate of salt-stress-related genes (TaNHX1, TaHKT1, and TaCAT1) compared with DHL2 and Sakha93 under salt stress conditions. Principle component analysis detection of the first two components explains 70.78% of the overall variation of all traits (28 out of 32 traits). A multiple linear regression model and path coefficient analysis showed a coefficient of determination (R2) of 0.93. The models identified two interpretive variables, number of spikelets, and/or number of kernels, which can be unbiased traits for assessing wheat DHLs under salinity stress conditions, given their contribution and direct impact on the grain yield.

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Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.

Publications

Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions.

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