Nuclear resonance vibrational spectroscopy (NRVS) is an emerging site-specific probe of active site vibrational dynamics in metalloproteins. 1,2 NRVS is a synchrotron-based technique that uniquely targets the vibrations of a Mössbauer nucleus, such as 57 Fe, without interference from vibrations of other atoms, and reveals not only the frequency, but also the (mean squared) amplitude, 1b,2a of all vibrations of the probe nucleus along the direction of the incident X-ray beam. Quantitative characterization of vibrational modes involving a reactive probe atom can illuminate mechanisms of complex biomolecules.Reactions with heme proteins mediate the physiological effects of nitric oxide (NO). The proposed trigger for activation of soluble guanylate cyclase (sGC) is rupture of the covalent Fe-His bond 3a,b in the heme-containing domain 3c,d upon NO binding to the Fe. A thermodynamic consequence of NO-induced weakening of a trans Fe-imidazole bond, as observed in several heme systems, is that imidazole binding should weaken a trans Fe-NO bond. Model compound structures support such a reciprocal negative trans interaction, 4a although protein structural data 4b-d may not have sufficient precision to resolve the 3 pm increase in Fe-NO bond length due to imidazole binding.On the other hand, vibrational frequencies respond sensitively to bond length changes of this magnitude, and it is therefore puzzling that the frequency attributed to stretching of the Fe-NO bond in six-coordinate imidazole-ligated heme proteins 5 is higher, rather than lower, than the frequencies observed for five-coordinate iron nitrosyl hemes. For example, the assigned Fe-NO stretching frequencies of the 5-and 6-coordinate NO complexes with myoglobin (MbNO) are 521 cm −1 and 552 cm −1 , respectively. 5c NRVS measurements on 6-coordinate MbNO suggest reexamination of this issue (Fig. 1A). The Fe-weighted vibrational density of states (VDOS) D(ν) samples the vibrational kinetic energy distribution (KED), with each mode contributing an area e Fe 2 equal to the fraction of mode energy associated with Fe motion. 2a,d The e Fe 2 = 0.11 area of the feature at 547 cm −1 is well below the e Fe 2 = 0.23-0.33 range that we observed for the FeNO stretching mode in a series of 5-coordinate nitrosyl porphyrins, 2d but is clearly visible because of the distinctly improved signal quality compared to previously published NRVS measurements on myoglobin. 1a,b,d In contrast, a mode with an area e Fe 2 = 0.25 appears at 443 cm −1 , near a

Recent advances in molecular biology such as gene editing [Mahas et al., 2018], bioelectric recording and manipulation [Levin, 2012a] and live cell microscopy using fluorescent reporters [Mutoh et al., 2012], [V. Sekar et al., 2011] -especially with the advent of light-controlled protein activation through optogenetics [Bugaj et al., 2017] have provided the tools to measure and manipulate molecular signaling pathways with unprecedented spatiotemporal precision. This has produced ever increasing detail about the molecular mechanisms underlying development and regeneration in biological organisms. However, an overarching concept -that can predict the emergence of form and the robust maintenance of complex anatomy -is largely missing in the field. Classic (i.e., dynamic systems and analytical mechanics) approaches such as least action principles are difficult to use when characterizing open, far-from equilibrium systems that predominate in Biology. Similar issues arise in neuroscience when trying to understand neuronal dynamics from first principles. In this (neurobiology) setting, a variational free energy principle has emerged based upon a formulation of self-organization in terms of (active) Bayesian inference. The free energy principle has recently been applied to biological self-organization beyond the neurosciences [Friston et al., 2015], . For biological processes that underwrite development or regeneration, the Bayesian inference framework treats cells as information processing agents, where the driving force behind morphogenesis is the maximization of a cell's model evidence. This is realized by the appropriate expression of receptors and other signals that correspond to the cell's internal (i.e., generative) model of what type of receptors and other signals it should express. The emerging field of the free energy principle in pattern formation provides an essential quantitative formalism for understanding cellular decision-making in the context of embryogenesis, regeneration, and cancer suppression. In this paper, we derive the mathematics behind Bayesian inference -as understood in this framework -and use simulations to show that the formalism can reproduce experimental, top-down manipulations of complex morphogenesis. First, we illustrate this 'first principle' approach to morphogenesis through simulated alterations of anterior-posterior axial polarity (i.e., the induction of two heads or two tails) as in planarian regeneration. Then, we consider aberrant signaling and functional behavior of a single cell within a cellular ensemble -as a first step in carcinogenesis as false 'beliefs' about what a cell should 'sense' and 'do'. We further show that simple modifications of the inference process can cause -and rescuemis-patterning of developmental and regenerative events without changing the implicit generative model of a cell as specified, for example, by its DNA. This formalism offers a new road map for understanding developmental change in evolution and for designing new interventions in regenerative med...

Green fluorescent protein (GFP) fluoresces efficiently under blue excitation despite major electrostatic rearrangements resulting from photoionization of the chromophore and neutralization of Glu-222. A competing phototransformation process, which ionizes the chromophore and decarboxylates Glu-222, mimics the electrostatic and structural changes in the fluorescence photocycle. Structural and spectroscopic analysis of the cryogenically stabilized photoproduct at 100 K and a structurally annealed intermediate of the phototransformed protein at 170 K reveals distinct structural relaxations involving protein, chromophore, solvent, and photogenerated CO 2 . Strong structural changes of the 100 K photoproduct after decarboxylation appear exclusively within 15 Å of the chromophore and include the electrostatically driven perturbations of Gln-69, Cys-70, and water molecules in an H-bonding network connecting the chromophore. X-ray crystallography to 1.85 Å resolution and static and picosecond time-resolved IR spectroscopy identify structural mechanisms common to phototransformation and to the fluorescence photocycle. In particular, the appearance of a 1697 cm ؊1 (؉) difference band in both photocycle and phototransformation intermediates is a spectroscopic signature for the structural perturbation of Gln-69. This is taken as evidence for an electrostatically driven dynamic response that is common to both photoreaction pathways. The interactions between the chromophore and the perturbed residues and solvent are decreased or removed in the T203H single and T203H/Q69L double mutants, resulting in a strong reduction of the fluorescence quantum yield. This suggests that the electrostatic response to the transient formation of a buried charge in the wild type is important for the bright fluorescence.Green fluorescent protein (GFP) 2 (1, 2) from Aequorea victoria is highly fluorescent with 400 nm excitation, despite major electrostatic rearrangements resulting from rapid charge transfer to the excited chromophore (3). Rapid excited state proton transfer (ESPT) (4 -6) follows excitation of the neutral, phenolic chromophore, and the resulting excited phenolate state I* exhibits high quantum efficiency redshifted emission at 508 nm, with a 3.0-ns lifetime (3). The mechanism by which the protein environment suppresses non-radiative processes remains unexplained. Proposals for the ESPT pathway include a hydrogen bonding network connecting the chromophore phenolic oxygen to Glu-222 via a water molecule and Ser-205 (7-9). Recently, the possibility of a proton transfer pathway including Glu-222, Asp-82, and Glu-5 was put forward (10). The transient infrared absorption reportedly developing at 1706 cm Ϫ1 with excitation of GFP in D 2 O (9) could not distinguish between these proposals. However, experimental evidence for the identity of the proton acceptor has recently been provided from comparison with the E222D mutant (11). Low quantum yield electron transfer from Glu-222 to the photoexcited chromophore triggers decarboxylation of the...