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

Abstract

Cytochrome c (cyt c) has long been utilized as a model system to study metalloprotein folding dynamics and the interplay between active site ligation and tertiary structure. However, recent reports regarding the weakness of the native Fe(II)-S bond (Fe-Met80) call into question the role of the active site ligation in the protein folding process. In order to investigate the interplay between protein conformation and active site structures, we directly tracked the evolution of both during a photolysis-induced folding reaction using X-ray transient absorption spectroscopy and time-resolved X-ray solution scattering techniques. We observe an intermediate Fe-Met80 species appearing on $2 ms timescale, which should not be sustained without stabilization from the folded protein structure. We also observe the appearance of a new active site intermediate: a weakly interacting Fe-H 2 O state. As both intermediates require stabilization of weak metal-ligand interactions, we surmise the existence of a local structure within the unfolded protein that protects and limits the movement of the ligands, similar to the entatic state found in the native cyt c fold. Furthermore, we observe that in some of the unfolded ensemble, the local stabilizing structure is lost, leading to expansion of the unfolded protein structure and misligation to His26/His33 residues.

Rapid 3-dimensional shape determination of globular proteins by mobility capillary electrophoresis and native mass spectrometry

“…However, the functions of a protein also depend on its stereostructure, which is not being provided by conventional MS based technologies. Biophysical methods such as X-ray crystallography, [4][5][6][7] nuclear magnetic resonance (NMR), [8][9][10] and electron microscopy [11][12][13] could provide detailed structural information of proteins but require laborious sample preparation, and the analysis of heterogeneous mixtures remains challenging. [14][15][16][17][18] Nanopores and nanopipettes have also been developed to analyze the volume, surface charge and structure dynamic process of proteins.…”

Resolving Dynamics in the Ensemble: Finding Paths through Intermediate States and Disordered Protein Structures

“…The X-ray scattering differences at longer time delays tracked the propagation of heme-doming motions into conformational changes in the outer protein, revealed in the species-associated difference signals in the SAXS regime. 60 A similar experiment on cyt c was also conducted to monitor structural changes upon electron transport, initiated by the photoexcitation of NADH. 63 Although this folding pathway did not reveal disordered intermediate states, the experimental data were modeled with an ensemble of MD simulated structures.…”

“…An obvious application of pump–probe approaches is toward photoactive proteins. For example, the structural dynamics of heme proteins (myoglobin and cytochrome c oxidase) have been widely studied with laser-triggered photolysis of a ligand to observe subsequent heme-doming and transient intermediates, critical in active site activation and oxidase reaction mechanisms. ,− Cytochrome c (cyt c ), a 104-residue electron transport chain protein, is a long-established model system for heme protein biophysics. In parallel time-resolved spectroscopy and scattering experiments, the folding mechanisms, including transient intermediates, of cyt c were observed upon the photodissociation of a CO-bound ground state.…”

“…(c) Species-associated difference (SAD) on the left and their corresponding relative measured population on the right for the two intermediates, U M and U H , and the final unfolded state, F M . Reproduced from ref with permission from the Royal Society of Chemistry.…”

“…Since the EXAFS structural characterization and wide-angle X-ray scattering (WAXS) were well correlated to describe the heme and nearby ligand dynamics, the two parallel observations were stitched together to distinguish more transient species than each experiment could individually. The X-ray scattering differences at longer time delays tracked the propagation of heme-doming motions into conformational changes in the outer protein, revealed in the species-associated difference signals in the SAXS regime . A similar experiment on cyt c was also conducted to monitor structural changes upon electron transport, initiated by the photoexcitation of NADH .…”