Real-Time Protein Unfolding: A Method for Determining the Kinetics of Native Protein Denaturation using a Quantitative Real-Time Thermocycler

Biggar, Kyle K.; Dawson, Neal J.; Storey, Kenneth B.

doi:10.2144/0000113922

Cited by 42 publications

(41 citation statements)

References 16 publications

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“…The T m of purified PumHD and Pumby variants was measured using a thermal shift assay with SYPRO Orange (Invitrogen) dye according to the previously described protocol (51). Briefly, the 2.5 μM peptide samples were prepared in 100 mM Hepes (pH 7.4), 150 mM NaCl and 5× SYPRO Orange dye.…”

Section: Methodsmentioning

confidence: 99%

Programmable RNA-binding protein composed of repeats of a single modular unit

Adamala

Martin-Alarcon

Boyden

2016

Proc. Natl. Acad. Sci. U.S.A.

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The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby-RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.

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Section: Methodsmentioning

confidence: 99%

Programmable RNA-binding protein composed of repeats of a single modular unit

Adamala

Martin-Alarcon

Boyden

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Cold-adapted enzymes are commercially valuable for bioremediation, textile and food production and as additives in detergents (Biggar et al 2012). In addition, these enzymes sometimes have unusual substrate specificity (Feller and Gerday 2003).…”

Section: Introductionmentioning

confidence: 99%

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

et al. 2015

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Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans.A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H.americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

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“…The differential scanning fluorimetry technique has recently provided a novel mechanism for determining temperature stability using a quantified real-time thermocycler (Niesen et al, 2007). Furthermore, a modification to this technique, whereby fluorescence is monitored during protein unfolding in the presence of increasing amounts of urea or other denaturants, allows for a more precise indication of changes to the proteins surface structure (Biggar et al, 2012). Using both DSF techniques in combination with kinetic analysis we aim to demonstrate the effectiveness of this technique in resolving the core and surface stability differences between two proteins with high sequence similarity: the 4H tetramer from M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT heart and the 4M tetramer of LDH from pig (Sus scrofa) tissues.…”

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confidence: 99%

“…SEM p<0.05, Student's t-test. Biggar et al, (2012) demonstrated the sensitivity of using a quantified real-time thermocycler to analyze protein unfolding using urea. H-LDH and M-LDH isozymes were analyzed as described in Biggar et al, (2012) with 1.25 mg/ml of protein being used.…”

mentioning

confidence: 99%

“…Biggar et al, (2012) demonstrated the sensitivity of using a quantified real-time thermocycler to analyze protein unfolding using urea. H-LDH and M-LDH isozymes were analyzed as described in Biggar et al, (2012) with 1.25 mg/ml of protein being used. SYPRO Orange dye (40X diluted; 2.5 µL) was mixed with 15.5 µL of increasing concentrations of urea prepared in DSF buffer in a 96-well PCR plate.…”

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confidence: 99%

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