Determining the Conformational Stability of a Protein from Urea and Thermal Unfolding Curves

Grimsley, Gerald R.; Treviño, Saul; Thurlkill, Richard L.; Scholtz, J. Martin

doi:10.1002/0471140864.ps2804s71

Cited by 17 publications

(14 citation statements)

References 30 publications

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“…The PC, PE-I and PE-II, displayed fluorescence emission maxima at 635, 569 and 562 nm, respectively. We then performed thermal unfolding curves for each of these 18 purified PBPs, in order to determine their mid-unfolding temperatures, T 50% , a commonly used proxy for assessing and comparing the thermostability and molecular flexibility of proteins ( Supplementary Figures S2 and S3; Jaenicke, 1991;Grimsley et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

“…Phycobiliprotein purification was carried out using sucrose gradients and isoelectric focusing, as previously described (Six et al, 2005). PBP structural thermostability was assessed by monitoring 5.5 ± 0.7 6.9 ± 0.9 5.9 ± 0.5 5.3 ± 0.6 3.1 ± 0.4 4.4 ± 0.4 Temperature range for growth (°C) 18 Phycobilisome thermoadaptation in Synechococcus J Pittera et al phycobilin absorbance and fluorescence along protein thermal unfolding curves (Grimsley et al, 2013), from 30°C to 85°C, with PBP solutions at~30 nM for PEs (2.15 × 10 6 M − 1 cm − 1 and 2.41 × 10 6 M − 1 cm − 1 at 545 nm for PE-II and PE-I, respectively; Glazer and Hixson, 1977;Wyman 1992) and 100 nM for PC (2 × 10 5 M − 1 cm − 1 at 620 nm as estimated from Glazer et al, 1973 andHixson, 1975). Absorption and fluorescence emission spectra were recorded during a progressive temperature increase, performed by 5°C steps of 5 min each.…”

Section: Phycobiliprotein Denaturation Curvesmentioning

confidence: 99%

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Adaptive thermostability of light-harvesting complexes in marine picocyanobacteria

2016

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Marine Synechococcus play a key role in global oceanic primary productivity. Their wide latitudinal distribution has been attributed to the occurrence of lineages adapted to distinct thermal niches, but the physiological and molecular bases of this ecotypic differentiation remain largely unknown. By comparing six strains isolated from different latitudes, we showed that the thermostability of their light-harvesting complexes, called phycobilisomes (PBS), varied according to the average sea surface temperature at strain isolation site. Comparative analyses of thermal unfolding curves of the three phycobiliproteins (PBP) constituting PBS rods suggested that the differences in thermostability observed on whole PBSs relied on the distinct molecular flexibility and stability of their individual components. Phycocyanin was the least thermostable of all rod PBP, constituting a fragility point of the PBS under heat stress. Amino-acid composition analyses and structural homology modeling notably revealed the occurrence of two amino-acid substitutions, which might have a role in the observed differential thermotolerance of this phycobiliprotein among temperature ecotypes. We hypothesize that marine Synechococcus ancestors occurred first in warm niches and that during the colonization of cold, high latitude thermal niches, their descendants have increased the molecular flexibility of PBP to maintain optimal light absorption capacities, this phenomenon likely resulting in a decreased stability of these proteins. This apparent thermoadaptability of marine Synechococcus has most probably contributed to the remarkable ubiquity of these picocyanobacteria in the ocean.

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Section: Resultsmentioning

confidence: 99%

Section: Phycobiliprotein Denaturation Curvesmentioning

confidence: 99%

Adaptive thermostability of light-harvesting complexes in marine picocyanobacteria

2016

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“…We then plotted the ΔH m against Tm to yield ΔCp (Table 2), which is the slope. Finally, the unfolding free energy change ΔG NU at 25°C is calculated using the modified Gibbs-Helmholtz equation according to Grimsley et al, (2013):

Δ G (T) = Δ H_{↓ m} (1 - T / T_{↓ m}) - Δ C_{↓ p} [(T_{↓ m} - T) + T l n (T / T_{↓ m})]

where T = 298.15K.…”

Section: Methodsmentioning

confidence: 99%

Super Spy variants implicate flexibility in chaperone action

Qin

Wang

Petrotchenko

et al. 2014

eLife

104

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Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function.DOI: http://dx.doi.org/10.7554/eLife.01584.001

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“…Thermodynamic Characterization of hASNase1 Conformational Stability-The conformational stability of hASNase1 and EcASNase1 was studied by monitoring changes in intrinsic fluorescence of the stepwise urea-denatured proteins (35,36). The final urea concentrations ranged from 0.5 to 8 M using a stock solution of 10 M urea dissolved in 50 mM Tris-Cl, 100 mM NaCl, pH 8.…”

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confidence: 99%

Human 60-kDa Lysophospholipase Contains an N-terminal l-Asparaginase Domain That Is Allosterically Regulated by l-Asparagine

Karamitros

Konrad

2014

Journal of Biological Chemistry

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Background:The 60-kDa human lysophospholipase comprises an N-terminal domain with predicted, yet uncharacterized L-asparaginase activity and a C-terminal ankyrin repeat-like domain. Results:The N-terminal domain, termed hASNase1, was identified as a functional structural unit possessing catalytic activity. Conclusion: hASNase1 is an allosterically regulated bacterial-type cytoplasmic L-asparaginase. Significance: Domains of multifunctional human proteins harbor homologs of prokaryotic enzymes displaying similar structural and kinetic features.

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Determining the Conformational Stability of a Protein from Urea and Thermal Unfolding Curves

Cited by 17 publications

References 30 publications

Adaptive thermostability of light-harvesting complexes in marine picocyanobacteria

Adaptive thermostability of light-harvesting complexes in marine picocyanobacteria

Super Spy variants implicate flexibility in chaperone action

Human 60-kDa Lysophospholipase Contains an N-terminal l-Asparaginase Domain That Is Allosterically Regulated by l-Asparagine

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