Circular dichroism spectroscopic assessment of structural changes upon protein thermal unfolding at contrasting pH: Comparison with molecular dynamics simulations
Por:
García-Gutiérrez P., Camarillo-Cadena M., Vera-Robles L.I., Zubillaga R.A., Hernández-Arana A.
Publicada:
1 ene 2022
Resumen:
In most instances, the usual fastness of protein unfolding events hinders determining changes in secondary structures associated with this process because these determinations rely on the recording of high-resolution circular dichroism (CD) spectra. In this work, far-UV CD spectra, recorded at ten-minute intervals, were used to evaluate the time course followed by four classes of secondary structures in the slow temperature-induced unfolding of yeast triosephosphate isomerase (yTIM) under distinct pH conditions. CONTIN-LL and SELCON3 algorithms were used for the deconvolution of spectra. Both algorithms furnished helix and unordered structure contents that changed according to first-order kinetics, agreeing with the behavior shown by CD data at specific wavelengths. Analyses of unfolded yTIM spectra, using a dataset that includes spectra of unfolded proteins and either one of the two algorithms, clearly showed a more unordered protein structure at high pH; this finding was corroborated with analysis of the difference spectra. Molecular dynamics (MD) simulations performed with AMBER and OPLS force fields resulted in more extensive loss of helices and gain in coils at high pH, in agreement with spectroscopic results. However, structural differences between low- and high-pH unfolded yTIM were relatively small. Comparison of results from CD and MD thus point to the need of fine-tuning of MD procedures. © 2022 Elsevier B.V.
Filiaciones:
Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Iztapalapa CDMX, 09340, Mexico
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