Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

Greenfield, Norma J.

doi:10.1038/nprot.2006.204

Cited by 758 publications

(835 citation statements)

References 44 publications

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“…We attempted to compare the thermal stability of C. trachomatis Serovars D, E, F, and J nMOMP as well as C. muridarum nMOMP by monitoring heat induced protein unfolding by CD spectroscopy 44. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

et al. 2018

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Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D–K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo‐trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR‐FTIR) reduction in β‐sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α‐helix content increased by 7% (CD) to 13% (ATIR‐FTIR). Maintenance of a native‐like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

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Section: Resultsmentioning

confidence: 99%

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

et al. 2018

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“…30 Contrary to the thermal inactivation profiles, the protein is directly studied during the actual melting process. Examples of denaturation experiments performed for LipA variants at pH 7 are shown in the Supporting Information (Fig.…”

Section: Protein Denaturation Studies Using CDmentioning

confidence: 99%

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

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Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

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“…Data were collected at 0.5°C intervals at a scan rate of 60°C per hour from 4°C to 90°C. The change in mean residue ellipticity, , as a function of temperature was modeled using a nonlinear least squares algorithm assuming the 2-state transition of a monomer from a folded to an unfolded state with a change in heat capacity, ⌬Cp, between the folded and unfolded forms (17). A detailed description of the calculations used is given in S3.…”

Section: Circular Dichroism Analysismentioning

confidence: 99%

“…WT and all 4 mutant proteins were modeled as a 2-state transition of monomer from a folded to an unfolded state with a change in heat capacity, ⌬Cp, between the folded and unfolded forms (17). The calculated thermal midpoints of unfolding for R1845W, E1886K, and H1901L are slightly decreased from WT, whereas L1793P displays a relatively larger decrease in thermal stability (Fig.…”

mentioning

confidence: 99%

Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

Armel

Leinwand

2009

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

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Myosin storage myopathy (MSM) is a congenital myopathy characterized by the presence of subsarcolemmal inclusions of myosin in the majority of type I muscle fibers, and has been linked to 4 mutations in the slow/cardiac muscle myosin, ␤-MyHC (MYH7). Although the majority of the >230 disease causing mutations in MYH7 are located in the globular head region of the molecule, those responsible for MSM are part of a subset of MYH7 mutations that are located in the ␣-helical coiled-coil tail. Mutations in the myosin head are thought to affect the ATPase and actin-binding properties of the molecule. To date, however, there are no reports of the molecular mechanism of pathogenesis for mutations in the rod region of muscle myosins. Here, we present analysis of 4 mutations responsible for MSM: L1793P, R1845W, E1886K, and H1901L. We show that each MSM mutation has a different molecular phenotype, suggesting that there are multiple mechanisms by which MSM can be caused. These mechanisms range from thermodynamic and functional irregularities of individual proteins (L1793P), to varying defects in the assembly and stability of filaments formed from the proteins (R1845W, E1886K, and H1901L). In addition to furthering our understanding of MSM, these observations provide the first insight into how mutations affect the rod region of muscle myosins, and provide a framework for future studies of disease-causing mutations in this region of the molecule.

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Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

Cited by 758 publications

References 44 publications

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

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