Methods for Protein Characterization by Mass Spectrometry, Thermal Shift (ThermoFluor) Assay, and Multiangle or Static Light Scattering

Nettleship, Joanne E.; Brown, James; Groves, Matthew R.; Geerlof, Arie

doi:10.1007/978-1-60327-058-8_19

Cited by 125 publications

(115 citation statements)

References 12 publications

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“…3A), characteristic of a protein containing 65% α-helix, 17% turns, and 8% unfolded, as calculated with DichroWeb (11) using two different algorithms, SELCON3 (12) and CONTIN (13). A ThermoFluor assay (14) was used to monitor thermal unfolding of αSyn, and to detect whether a hydrophobic core is present in the oligomer, as determined by an increase in fluorescence emitted by the dye present. We observed a sigmoidal unfolding curve for the αSyn construct, indicating a cooperative unfolding with exposure of hydrophobic residues (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

A soluble α-synuclein construct forms a dynamic tetramer

Wang

Perovic

Chittuluru

et al. 2011

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

416

519

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A heterologously expressed form of the human Parkinson diseaseassociated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 Nterminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three-or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. T he protein α-synuclein (αSyn) is associated with the two most prevalent neurodegenerative diseases, Parkinson disease (PD) and Alzheimer's disease (AD). The presence of αSyn-rich aggregates (Lewy bodies) in neurons of the substantia nigra is the defining histopathological hallmark of PD, and is used to differentiate PD from other neurological disorders (1). Monogenic point mutations (A30P, A53T, and E46K) as well as gene duplication and triplication of the αSyn locus have been identified as causal factors of early onset familial PD; E46K has also been associated with Lewy body dementia, the second most common form of dementia after AD (2-4).αSyn is small (140 residues), and though the C-terminal region (∼residues 100-140) is highly acidic and expected to be disordered, the first 100 residues are predicted to be structured and to have α-helical propensity (SI Appendix, Fig. S1). Stable helical structures have been detected by circular dichroism (CD) and NMR when αSyn is incubated with detergent micelles and lipid vesicles (5, 6). Soluble αSyn is typically referred to as an "intrinsically disordered" protein (7,8). However, we herein report the biophysical characterization of a purified soluble form of αSyn that is oligomeric and fractionally occupies helical structures in the absence of micelles or vesicles. The αSyn construct used in our work is purified by use of an N-terminal GST affinity tag under mild conditions to preserve any native structure. After removal of the GST tag, a 10-residue N-terminal extension remains on the αSyn. However, the similarity of the 1 H, 15 N heteronuclear single-quantum coherence (HSQC) fingerprint of our αSyn construct (SI Appendix, Figs. S2 and S3) to those reported by other groups for αSyn suggests that the N-terminal extension does not change structural tendencies significantly. The αSyn construct described here is not toxic to membranes or cells, does not readily aggregate or ...

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Section: Resultsmentioning

confidence: 99%

A soluble α-synuclein construct forms a dynamic tetramer

Wang

Perovic

Chittuluru

et al. 2011

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

416

519

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“…The thermal stabilities of wild-type hSR and the C217S, K221E, and S84G/P111L mutants were assessed using ThermoFluor, a biophysical technique for determination of relative protein stabilities based on fluorophore partition into the denatured protein upon temperature increase [28][29][30] . In the absence of SR activators, wild-type, C217S, and K221E hSR exhibited melting temperatures of 63.2 ± 0.53, 63.1 ± 0.17, and 62.7 ± 0.32°C, respectively.…”

Section: Thermal Stability Of C217s K221e and S84g/p111l Hsrmentioning

confidence: 99%

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Hoffman

Jirásková

Zvelebil

et al. 2010

Collect. Czech. Chem. Commun.

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Human serine racemase (hSR) is a cytosolic pyridoxal-5′-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer's disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR's promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structurally and functionally important residues. In particular, we identify S84 and P111 as residues crucial for hSR activity and C217 and K221 as residues important for binding of the Mg 2+ cofactor as well as for overall stability of the enzyme. Keywords: D-Serine; Serine racemase; Error-prone PCR; Random mutagenesis; ThermoFluor assay; Enzymes; Racemases; Enzyme engineering; In vitro evolution.Eukaryotic serine racemase (SR; EC 5.1.1.18) is a cytosolic pyridoxal-5′-phosphate (PLP) dependent enzyme responsible for the production of D-serine from L-serine and vice versa. In the mammalian central nervous system (CNS), D-serine acts as an endogenous coagonist of N-methyl-D-aspartate (NMDA) receptors, which are involved in glutamate-mediated excitatory neurotrans-

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“…Binding of a Phosphate Group in Solution-We carried out DSF and CD assays (54) to investigate the binding of a phosphate in solution to the KinB-SD. The protein unfolding temperature (T m ) was measured using DSF at different phosphate concentrations.…”

Section: Resultsmentioning

confidence: 99%

Sensor Domain of Histidine Kinase KinB of Pseudomonas

Tan

Chhor

Binkowski

et al. 2014

Journal of Biological Chemistry

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Background:The sensor domain (SD) of histidine kinase (HK) KinB (KinB-SD) receives signals from the environment and induces a transduction cascade. Results: Structures of the KinB-SD were obtained in ligand-free, phosphate-bound, and mutant forms. Conclusion:The unique helix-swapped KinB-SD structure forms a ligand-binding cavity on the dimer interface. Significance: KinB-SD studies provide insights into the signal transduction and identity of potential signaling molecules.

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Methods for Protein Characterization by Mass Spectrometry, Thermal Shift (ThermoFluor) Assay, and Multiangle or Static Light Scattering

Cited by 125 publications

References 12 publications

A soluble α-synuclein construct forms a dynamic tetramer

A soluble α-synuclein construct forms a dynamic tetramer

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Sensor Domain of Histidine Kinase KinB of Pseudomonas

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