Robust and convenient analysis of protein thermal and chemical stability

Niklasson, Markus; Andrésen, Cecilia; Helander, Sara; Roth, Marie; Kahlin, Anna Zimdahl; Appell, Malin Lindqvist; Mårtensson, Lars-Göran; Lundström, Patrik

doi:10.1002/pro.2809

Cited by 56 publications

(56 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As reported previously, CLD N-lobe undergoes two-state unfolding with denaturation midpoint temperatures of 64.4 ± 0.4°C and 90 ± 3°C for CLD N-lobe apo and CLD N-lobe Ca , respectively [17]. In contrast, the thermal denaturation profiles for several samples of CLD C-lobe Ca were not compatible with two-state denaturation (see inset of Fig 2C and S1 Fig) and it was not fruitful to estimate the midpoint temperature of denaturation.…”

Section: Resultsmentioning

confidence: 63%

Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

et al. 2017

Self Cite

View full text Add to dashboard Cite

Calcium dependent protein kinases are unique to plants and certain parasites and comprise an N-terminal segment and a kinase domain that is regulated by a C-terminal calcium binding domain. Since the proteins are not found in man they are potential drug targets. We have characterized the calcium binding lobes of the regulatory domain of calcium dependent protein kinase 3 from the malaria parasite Plasmodium falciparum. Despite being structurally similar, the two lobes differ in several other regards. While the monomeric N-terminal lobe changes its structure in response to calcium binding and shows global dynamics on the sub-millisecond time-scale both in its apo and calcium bound states, the C-terminal lobe could not be prepared calcium-free and forms dimers in solution. If our results can be generalized to the full-length protein, they suggest that the C-terminal lobe is calcium bound even at basal levels and that activation is caused by the structural reorganization associated with binding of a single calcium ion to the N-terminal lobe.

show abstract

Section: Resultsmentioning

confidence: 63%

Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…At least three replicates were performed for each technique. Data were analyzed according to a two-state model and fitted using CD Pal [ 40 ]. The unfolding transitions were irreversible, impeding further thermodynamic analysis, and only the apparent midpoint unfolding temperature (T m app ) is reported.…”

Section: Methodsmentioning

confidence: 99%

Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients

Ribeiro

Gomes

Henriques

2020

IJMS

View full text Add to dashboard Cite

Riboflavin is the biological precursor of two important flavin cofactors—flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)—that are critical prosthetic groups in several redox enzymes. While dietary supplementation with riboflavin is a recognized support therapy in several inborn errors of metabolism, it has yet unproven benefits in several other pathologies affecting flavoproteins. This is the case for glutaric aciduria type I (GA-I), a rare neurometabolic disorder associated with mutations in the GCDH gene, which encodes for glutaryl-coenzyme A (CoA) dehydrogenase (GCDH). Although there are a few reported clinical cases that have responded to riboflavin intake, there is still not enough molecular evidence supporting therapeutic recommendation. Hence, it is necessary to elucidate the molecular basis in favor of riboflavin supplementation in GA-I patients. Here, using a combination of biochemical and biophysical methodologies, we investigate the clinical variant GCDH-p.Val400Met as a model for a phenotype associated with severe deflavinylation. Through a systematic analysis, we establish that recombinant human GCDH-p.Val400Met is expressed in a nonfunctional apo form, which is mainly monomeric rather than tetrameric. However, we show that exogenous FAD is a driver for structural reorganization of the mutant enzyme with concomitant functional recovery, improved thermolability, and resistance to trypsin digestion. Overall, these results establish proof of principle for the beneficial effects of riboflavin supplementation in GA-I patients.

show abstract

“…The software CDpal was used to analyze thermal denaturation data in order to obtain information on protein stability23. The two-state model and Autofit procedure were chosen to determine the melting temperature (Tm) of each protein samples.…”

Section: Methodsmentioning

confidence: 99%

Discovery of tauroursodeoxycholic acid biotransformation enzymes from the gut microbiome of black bears using metagenomics

Song

Wang

Tan

et al. 2017

Sci Rep

View full text Add to dashboard Cite

Tauroursodeoxycholic acid (TUDCA) has been used to treat many diseases effectively. 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) are two key enzymes that drive the efficient biosynthesis of TUDCA from taurochenodeoxycholic acid (TCDCA) in vitro. In this study, a metagenomic approach was used to isolate 7α- and 7β-HSDHs from fecal samples of black bears. Five new 7α-HSDHs and one new 7β-HSDH enzyme were discovered and identified from the gut microbiota of black bears, and four of them presented good enzymatic properties. Our data also suggest cooperation in the biotransformation of TUDCA by the gut microbiota in black bears. In conclusion, this work expands the natural enzyme bank of HSDHs, provides promising candidate enzymes for application in the biosynthesis TUDCA and the epimerization reaction of bile acids at the C-7 position, and provides a data set for the discovery of novel enzymes in the gut micriobiome of black bears.

show abstract

Robust and convenient analysis of protein thermal and chemical stability

Cited by 56 publications

References 20 publications

Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

Biophysical characterization of the calmodulin-like domain of Plasmodium falciparum calcium dependent protein kinase 3

Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation—Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients

Discovery of tauroursodeoxycholic acid biotransformation enzymes from the gut microbiome of black bears using metagenomics

Contact Info

Product

Resources

About