Relationship between the wavelength maximum of a protein and the temperature dependence of its intrinsic tryptophan fluorescence intensity

Saini, Komal; Deep, Shashank

doi:10.1007/s00249-010-0601-3

Cited by 12 publications

(5 citation statements)

References 17 publications

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“…We have modelled this curvature as a quadratic term, as previously suggested in the literature [22]. The fits of the three normalized datasets to Eq.…”

Section: Resultsmentioning

confidence: 99%

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Global analysis of protein stability by temperature and chemical denaturation

Hamborg

Horsted

Johansson

et al. 2020

Analytical Biochemistry

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The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5 -1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 µl is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 µg of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook.

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“…We have modelled this curvature as a quadratic term, as previously suggested in the literature [22]. The fits of the three normalized datasets to Eq.…”

Section: Resultsmentioning

confidence: 99%

“…The temperature dependence of the fluorescence signals of the native state is often linear whereas that of the denatured state is curved [21] and often may be described by adding a quadratic term [22], though other non-linear functions have also been used [23]. Thus we have:…”

Section: Temperature Denaturationmentioning

confidence: 99%

Global analysis of protein stability by temperature and chemical denaturation

Hamborg

Horsted

Johansson

et al. 2020

Analytical Biochemistry

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“…This apparent kink in the plot observed between 330 K and 350 K is in agreement with the reversible denaturation temperature determined by calorimetry and fluorescence measurements. 55,56 Likewise, the MSD of lysozyme hydrogen atoms in 0.5 M trehalose solution was calculated, wherein a decrease in the magnitude of the MSD could be seen (ESI, † Fig. S2), in line with the reduction of protein internal dynamics.…”

Section: Effect Of Trehalose On Protein Dynamicsmentioning

confidence: 98%

Revisiting the conundrum of trehalose stabilization

Katyal

Deep

2014

Phys. Chem. Chem. Phys.

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Protein aggregation and loss of protein's biological functionality are manifestations of protein instability. Cosolvents, in particular trehalose, are widely accepted antidotes against such destabilization. Although numerous theories have been promulgated in the literature with regard to its mechanism of stabilization, the present scenario is still elusive in view of the discrepancies existing in them. To this end, we have revisited the conundrum and attempted to rationalize the mechanism by conducting thorough investigation of the effect of trehalose on the native, partially unfolded and denatured states of protein "Lysozyme" by means of molecular dynamic (MD) simulations under different temperature and concentration regimes. Two-dimensional contour plots along with principal component analysis suggest that trehalose molecules offer on-pathway stabilization unaltering the principal direction of protein's motion, although it slows down protein dynamics so that the protein gets trapped in the homogeneous ensemble of conformations closer to the native state. Free energy landscape reveals higher population of native compared to intermediate and denatured states. Delphi results and calculation of the preferential interaction parameter demonstrate that this relative stabilization of the native state can be ascribed to be the consequence of favourable interactions of trehalose with side chains of certain loci on the protein surface encompassing polar flexible residues. Stability of protein results from the observed difference in binding affinity of trehalose for native and denatured states of protein. Our findings are at variance with the common conception of relative destabilization of the denatured state. Rather, we provide evidence for relative stabilization of the native state. This stabilization is due to interplay of protein-trehalose, water-trehalose, water-water, protein-water and trehalose-trehalose interactions.

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“…( 44) should perhaps be thought of as an empirical relationship that may in some cases describe the temperature dependence of fluorescence intensities (Lindorff-Larsen and Winther, 2001). Indeed, another possibility is to use a simple parabolic model to capture non-linear effects (Saini and Deep, 2010;Hamborg et al, 2020).…”

Section: K As a Function Of Temperaturementioning

confidence: 99%

Linking thermodynamics and measurements of protein stability

Lindorff‐Larsen¹,

Teilum²

2020

Preprint

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We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding. The primary focus is on the thermodynamics of reversible folding/unfolding transitions and the experimental methods that are available for extracting thermodynamic parameters. We highlight the importance of modelling both how the folding equilibrium depends on a perturbing variable such as temperature or denaturant concentration, and the importance of modelling the baselines in the experimental observables.

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Relationship between the wavelength maximum of a protein and the temperature dependence of its intrinsic tryptophan fluorescence intensity

Cited by 12 publications

References 17 publications

Global analysis of protein stability by temperature and chemical denaturation

Global analysis of protein stability by temperature and chemical denaturation

Revisiting the conundrum of trehalose stabilization

Linking thermodynamics and measurements of protein stability

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