A billion years of evolution manifest in nanosecond protein dynamics

Abstract

Protein dynamics form a critical bridge between protein structure and function, yet the impact of evolution on ultrafast processes inside proteins remains enigmatic. This study delves deep into nanosecond-scale protein dynamics of a structurally and functionally conserved protein across species separated by almost a billion years, investigating ten homologs in complex with their ligand. By inducing a photo-triggered destabilization of the ligand inside the binding pocket, we resolved distinct kinetic footprints for each homolog via transient infrared spectroscopy. Strikingly, we found a cascade of rearrangements within the protein complex which manifest in time points of increased dynamic activity conserved over hundreds of millions of years within a narrow window. Among these processes, one displays a subtle temporal shift correlating with evolutionary divergence, suggesting reduced selective pressure in the past. Our study not only uncovers the impact of evolution on molecular processes in a specific case, but has also the potential to initiate a field of scientific inquiry within molecular paleontology, where species are compared and classified based on the rapid pace of protein dynamic processes; a field which connects the shortest conceivable time scale in living matter (10[Formula: see text] s) with the largest ones (10[Formula: see text] s).

Keywords: biophysics; evolution; photoswitch; protein dynamics; transient infrared spectroscopy.

Fig. 1.

Structure and function of MCL-1 are conserved. (A) NMR structure of MCL-1 (gray) complexed with PUMA BH3 (yellow) (PDB: 2roc) (33). (B) Phylogeny of ten species whose MCL-homologs were selected for this study. The phylogeny and the corresponding evolutionary divergence time in million years (Ma) were taken from TimeTree5 and cover the current state of science (July 2023) (34). (C) Sequence identity of all investigated MCL-1 homologs (compared to H. sapiens) against evolutionary divergence time of the corresponding species. (D and E) Structural similarity between MCL-1 homologs (compared to H. sapiens), predicted with AlphaFold (35) (blue) and RosettaFold (36) (red). (F) MCL-1 homolog binding free energy for the PUMA BH3 peptide, plotted against the evolutionary divergence time. Yellow, linear fit $\pm$ SD. The Pearson correlation coefficient $r=0.39$ indicates that the binding free energy correlates weakly with evolutionary divergence time.

Fig. 2.

The conservation of ultrafast protein dynamics in MCL-1. (A) The protein MCL-1 in complex with the photoswitchable PUMA BH3 peptide. (B) Transient infrared spectroscopy of the photo-perturbed MCL-1/PUMA BH3 complex results in kinetic footprints for all homologs, exemplarily displayed in (C) for Mus musculus. The symbols serve as reference points for explanations in the main text. (D) Three dominating phases of increased dynamic activity are assessed (early-, mid-, late phase; dashed lines). Global multiexponential fitting with three time constants yields fits (red/blue) that cover the raw data (gray) well. Evolution-associated difference spectra (48, 49) (lower panel) were calculated for state S₁ (red), S₂ (yellow), S₃ (green), and for S_t (blue) with time constants ${\tau }_{\mathit{early}}$, ${\tau }_{\mathit{mid}}$, and ${\tau }_{\mathit{late}}$. (E) The difference spectra of all homologs display a high degree of similarity.

Fig. 3.

Time constants of increased dynamic activity ${\tau }_{\mathit{early}}$, ${\tau }_{\mathit{mid}}$, and ${\tau }_{\mathit{late}}$ against (A) evolutionary divergence in million years, Ma, and against (B) MCL-1’s affinity for PUMA BH3. Data in (A) and (B) are displayed with linear fits $\pm$ SD (yellow) and correlation coefficients $r$ (Pearson).

Fig. 4.

Isotope labeling helped to separate the signal contribution of MCL-1 and PUMA BH3 spatially and temporally. (A) Transient infrared spectroscopy with isotope-labeled MCL-1/PUMA BH3 complexes. From ten homologs, M. musculus was chosen as a representative, as ¹³C¹⁵N-labeling is highly cost intensive. Kinetic footprints of unlabeled samples (Upper panel), and of samples with ¹³C¹⁵N-labeled MCL-1 (Lower panel). The early protein response (triangle) is not shifted for the labeled sample. The mid protein response (circle) manifests in a distinct sharp feature that shifts from 1,660 to 1,610 cm⁻¹ upon isotope labeling (dashed lines). Isotope labeling did not separate any spectral features at the late phase of the protein response. (B) Evolution-associated difference spectra (see Data analysis in the Materials and Methods) display a relatively sharp positive band in state S₃ and S₄ (circle). In the nonlabeled complex, the sharp maximum coincides with the broad positive band of the blue shift. For the labeled complex, it is shifted by 50 cm⁻¹ from 1,660 to 1,610 cm⁻¹, as expected for ¹³C¹⁵N-labeling (53). (C) The time constants were assigned to dynamic processes in the protein complex (schematic overview).