Probing Cytochrome c Folding Transitions upon Phototriggered Environmental Perturbations Using Time-Resolved X-ray Scattering

Abstract

Direct tracking of protein structural dynamics during folding-unfolding processes is important for understanding the roles of hierarchic structural factors in the formation of functional proteins. Using cytochrome c (cyt c) as a platform, we investigated its structural dynamics during folding processes triggered by local environmental changes (i.e., pH or heme iron center oxidation/spin/ligation states) with time-resolved X-ray solution scattering measurements. Starting from partially unfolded cyt c, a sudden pH drop initiated by light excitation of a photoacid caused a structural contraction in microseconds, followed by active site restructuring and unfolding in milliseconds. In contrast, the reduction of iron in the heme via photoinduced electron transfer did not affect conformational stability at short timescales (<1 ms), despite active site coordination geometry changes. These results demonstrate how different environmental perturbations can change the nature of interaction between the active site and protein conformation, even within the same metalloprotein, which will subsequently affect the folding structural dynamics.

X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding

“…34,58 Further unfolding of U H species compared to U CO is in line with the expanded denatured state assumed by oxidized cyt c with HisX ligation in mild denaturing conditions, which has a R g of $30Å. 37,59 The assignment of the U H signal to unfolding is further corroborated by BIFT analysis, which indicates the loss of electron density at distances spanning up to maximum dimension D max of U CO (see ESI for details †).…”

“…The scattering difference signals free of the buffer heating contribution were obtained by using standard procedures as in previous works (see ESI for details †). [34][35][36][37] The resulting difference signals containing protein only contributions at selected time delays are shown in Fig. 2b.…”

Revealing Fast Structural Dynamics in pH-Responsive Peptides with Time-Resolved X-ray Scattering

“…In addition, TRXSS experiments are typically less intrusive to the protein sample as they do not require deuteration of the protein that may impact thermodynamics of the system. 22 We have previously shown that TRXSS provides a structural probe that, in conjunction with photoinduced pH-jumps, provides insights into dynamics of nonphotoactive systems on timescales of μs to ms. 23 Here, we show that TRXSS coupled with pH-jumps is a viable method for observing dynamics of intra- and intermolecular dynamics on nanosecond timescales.…”

Resolving structures of transition metal complex reaction intermediates with femtosecond EXAFS

“…Since the scattering signal consists, due to the lack of element specificity, of contributions from all atoms in the sample, disentangling the numerous inter-and intra-nuclear degrees of freedom involved in photoinduced structural dynamics can be troublesome. [23][24][25][26] Time-resolved EXAFS with B70 ps resolution is to date measured routinely at synchrotrons with numerous applications to investigate a broad range of materials. While the aforementioned XANES measurements of photoexcited TiO 2 revealed the timescale of the electron trapping, the extension to the EXAFS regime to study ZnO nanoparticles allowed the precise characterization of the hole trapping site as a singly charged oxygen vacancies.…”