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. 2023 Feb 9;14(5):1133-1139.
doi: 10.1021/acs.jpclett.2c03680. Epub 2023 Jan 27.

The Role of Transient Intermediate Structures in the Unfolding of the Trp-Cage Fast-Folding Protein: Generating Ensembles from Time-Resolved X-ray Solution Scattering with Genetic Algorithms

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The Role of Transient Intermediate Structures in the Unfolding of the Trp-Cage Fast-Folding Protein: Generating Ensembles from Time-Resolved X-ray Solution Scattering with Genetic Algorithms

Arnold M Chan et al. J Phys Chem Lett. .

Abstract

The Trp-cage miniprotein is one of the smallest systems to exhibit a stable secondary structure and fast-folding dynamics, serving as an apt model system to study transient intermediates with both experimental and computational analyses. Previous spectroscopic characterizations that have been done on Trp-cage have inferred a single stable intermediate on a pathway from folded to unfolded basins. We aim to bridge the understanding of Trp-cage structural folding dynamics on microsecond-time scales, by utilizing time-resolved X-ray solution scattering to probe the temperature-induced unfolding pathway. Our results indicate the formation of a conformationally extended intermediate on the time scale of 1 μs, which undergoes complete unfolding within 5 μs. We further investigated the atomistic structural details of the unfolding pathway using a genetic algorithm to generate ensemble model fits to the scattering profiles. This analysis paves the way for direct benchmarking of theoretical models of protein folding ensembles produced with molecular dynamics simulations.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Temperature series of equilibrium small-angle X-ray solution scattering pattern, S(q), of Trp-cage from 15 to 70 °C. The smoothed species associated scattering patterns for the fully folded and unfolded states are respectively shown in blue and red dashed lines. (Inset) relative population fractions of the equilibrium folded (F) and unfolded (Ust) species-associated protein scattering signals.
Figure 2.
Figure 2.
TRXSS time series and analysis results of Trp-cage unfolding. (a) Time series of scattering difference profiles from 300 ns to 10 μs. (b) Integrated difference signal at each time delay plotted with kinetic model from global analysis. (c) Species-associated population dynamics of the intermediate (I) and unfolded (U) states. (d) Species-associated scattering differences corresponding to the kinetic model from global analysis with comparison of unfolded species-associated difference profile (Ust) from static temperature series.
Figure 3.
Figure 3.
Kinetically observed unfolding states. (a) Species associated reconstructed SAXS patterns corresponding to each state. (b) Pair distribution function of each state.
Figure 4.
Figure 4.
Histograms of Rg for genetic algorithm results of reconstructed ensemble species and linear combination fit results of reconstructed experimental species associated scattering patterns. (a–c) Rg histograms of the average genetic algorithm results for each species associated scattering pattern with standard errors: F, I, and U, respectively. (d–f) Linear combination fit results for clustered representative structures for each species (F, I, and U, respectively); average theoretical ensemble scattering shown in black; experimentally reconstructed species associated scattering signal shown in gray.
Figure 5.
Figure 5.
Conformational heterogeneity of each state shown by structural model fitting. (a) Representative conformations of Trp-cage obtained from structural clustering shown with associated species ensemble population fraction from linear combination fitting. (b) Structural similarities between extracted conformations shown by Rg comparison plot. Experimentally observed ensemble Rg plotted as gray diamonds.

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