We present BindFlow, a Python-based software for automated absolute binding free energy (ABFE) calculations at the free energy perturbation (FEP) or at the molecular mechanics Poisson-Boltzmann/generalized Born surface area [MM(PB/GB)SA] level of theory. BindFlow is free, open-source, user-friendly, easily customizable, runs on workstations or distributed computing platforms, and provides extensive documentation and tutorials. BindFlow uses GROMACS as molecular dynamics engine and provides built-in support for the small-molecule force fields GAFF, OpenFF, and Espaloma. We test BindFlow by computing affinities for 139 ligand/target pairs, involving eight different targets including six soluble proteins, one membrane protein and one non-protein host–guest system. Quantified by Pearson, Kendall, and Spearman correlations coefficients, we find that the agreement of BindFlow predictions with experiments are overall similar to gold standards in the field. Interestingly, we find that MM(PB/GB)SA achieves correlations that, for some systems and force fields, approach those obtained with FEP, while requiring only a fraction of the computational cost. This study establishes BindFlow as a validated and accessible tool for ABFE calculations.

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms. A large photoreceptor subfamily uses vitamin B₁₂ derivatives for light sensing, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions. The molecular mechanism of B₁₂ photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B₁₂ photoreceptor CarH from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co–C bond triggers a pathway of structural changes that propagate throughout CarH from the B₁₂ chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B₁₂ enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B₁₂-dependent photoreceptors for future optogenetic applications.

The hydration shell is an integral part of proteins since it plays key roles in conformational transitions, molecular recognition, and enzymatic activity. While the dynamics of the hydration shell have been described by spectroscopic techniques, the structure of the hydration shell remains less understood due to the lack of hydration shell-sensitive structural probes with high spatial resolution. We combined temperature-ramp small-angle X-ray scattering (T-ramp SAXS) from 255 to 335 K with molecular simulations to demonstrate that the hydration shells of the IgG-binding domain of Protein G (GB3) and the villin headpiece are remarkably temperature-sensitive. For proteins in the folded state, T-ramp SAXS data and explicit-solvent SAXS predictions consistently demonstrate decays of protein contrasts and radii of gyration with increasing temperature, which are shown to reflect predominantly temperature-sensitive, depleting hydration shells. The depletion is caused not merely by enhanced disorder within the hydration shells but also by partial displacements of surface-coordinated water molecules. Together, T-ramp SAXS and explicit-solvent SAXS calculations provide a novel structural view of the protein hydration shell, which underlies temperature-dependent processes such as cold denaturation, thermophoresis, or biomolecular phase separation.