Consensus model of a cyanobacterial light-dependent protochlorophyllide oxidoreductase in its pigment-free apo-form and photoactive ternary complex.

Photosynthetic organisms employ two different enzymes for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide), yielding the chlorophyll precursor chlorophyllide. First, a nitrogenase-like, light-independent (dark-operative) Pchlide oxidoreductase and secondly, a light-dependent Pchlide oxidoreductase (LPOR). For the latter enzyme, despite decades of research, no structural information is available. Here, we use protein structure modelling, molecular dynamics (MD) simulations combined with multi-wavelength analytical ultracentrifugation (MWA-AUC) and small angle X-ray scattering (SAXS) experiments to derive a consensus model of the LPOR apoprotein and the substrate/cofactor/LPOR ternary complex. MWA-AUC and SAXS experiments independently demonstrate that the apoprotein is monomeric, while ternary complex formation induces dimerization. SAXS-guided modelling studies provide a full-length model of the apoprotein and suggest a tentative mode of dimerization for the LPOR ternary complex, supported by published cross-link constraints. Our study provides a first impression of the LPOR structural organization.

Ternary Complex Formation and Photoactivation of a Photoenzyme Results in Altered Protein Dynamics.

The interplay between protein dynamics and catalysis remains a fundamental question in enzymology. We here investigate the ns-timescale dynamics of a light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), a photoenzyme crucial for chlorophyll synthesis. LPORs catalyze the light-triggered trans addition of a hydride and a proton across the C17═C18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). Because of the lack of an LPOR structure, the global structural and dynamic consequences of LPOR/Pchlide/NADPH ternary complex formation remain elusive. Moreover, photoactivation of LPORs by low-light preillumination is controversially discussed as unequivocal proof for this phenomenon is lacking. By employing quasielastic neutron spectroscopy (QENS), we show that the formation of the ternary holoprotein complex as well as photoactivation lead to progressive rigidification of the protein. These findings are supported by thermostability measurements, which reveal different melting behavior and thermostabilities for the apo- and holoprotein ternary complexes. Molecular dynamics simulations in good agreement with the experimental QENS results suggest that the increased flexibility observed for the apoprotein stems from structural fluctuations of the NADPH and Pchlide substrate binding sites of the enzyme. On the basis of our results, in conjunction with activity and stability measurements, we provide independent proof for LPOR photoactivation, defined as a process that modifies the protein structure and dynamics, resulting in an increased substrate turnover. Our findings advance the structural and dynamic understanding of LPORs and provide a first link between protein dynamics and catalysis for this enzyme class.

Small-angle X-ray scattering study of the kinetics of light-dark transition in a LOV protein.

Light, oxygen, voltage (LOV) photoreceptors consist of conserved photo-responsive domains in bacteria, archaea, plants and fungi, and detect blue-light via a flavin cofactor. We investigated the blue-light induced conformational transition of the dimeric photoreceptor PpSB1-LOV-R66I from Pseudomonas putida in solution by using small-angle X-ray scattering (SAXS). SAXS experiments of the fully populated light- and dark-states under steady-state conditions revealed significant structural differences between the two states that are in agreement with the known structures determined by crystallography. We followed the transition from the light- to the dark-state by using SAXS measurements in real-time. A two-state model based on the light- and dark-state conformations could describe the measured time-course SAXS data with a relaxation time τREC of ~ 34 to 35 min being larger than the recovery time found with UV/vis spectroscopy. Unlike the flavin chromophore-based UV/vis method that is sensitive to the local chromophore environment in flavoproteins, SAXS-based assay depends on protein conformational changes and provides with an alternative to measure the recovery kinetics.

Dynamics-stability relationships in apo- and holomyoglobin: a combined neutron scattering and molecular dynamics simulations study.

The removal of the heme group from myoglobin (Mb) results in a destabilization of the protein structure. The dynamic basis of the destabilization was followed by comparative measurements on holo- (holo-Mb) and apomyoglobin (apo-Mb). Mean-squared displacements (MSD) and protein resilience on the picosecond-to-nanosecond timescale were measured by elastic incoherent neutron scattering. Differences in thermodynamic parameters, MSD, and resilience were observed for both proteins. The resilience of holo-Mb was significantly lower than that of apo-Mb, indicating entropic stabilization by a higher degree of conformational sampling in the heme-bound folded protein. Molecular dynamics simulations provided site-specific information. Averaged over the whole structure, the molecular dynamics simulations yielded similar MSD and resilience values for the two proteins. The mobility of residues around the heme group in holo-Mb showed a smaller MSD and higher resilience compared to the same residue group in apo-Mb. It is of interest that in holo-Mb, higher MSD values are observed for the residues outside the heme pocket, indicating an entropic contribution to protein stabilization by heme binding, which is in agreement with experimental results.

Macromolecular dynamics in red blood cells investigated using neutron spectroscopy.

We present neutron scattering measurements on the dynamics of haemoglobin (Hb) in human red blood cells (RBCs) in vivo. Global and internal Hb dynamics were measured in the ps to ns time and Å length scales using quasi-elastic neutron backscattering spectroscopy. We observed the cross over from global Hb short-time to long-time self-diffusion. Both short- and long-time diffusion coefficients agree quantitatively with predicted values from the hydrodynamic theory of non-charged hard-sphere suspensions when a bound water fraction of around 0.23 gram H(2)O per gram Hb is taken into account. The higher amount of water in the cells facilitates internal protein fluctuations in the ps time scale when compared with fully hydrated Hb powder. Slower internal dynamics of Hb in RBCs in the ns time range were found to be rather similar to results obtained with fully hydrated protein powders, solutions and Escherichia coli cells.

New insights of membrane environment effects on MscL channel mechanics from theoretical approaches.

The prokaryotic mechanosensitive channel of large conductance (MscL) is a remarkable integral membrane protein. During hypo-osmotic shock, it responses to membrane tension through large conformational changes, that lead to an open state of the pore. The structure of the channel from Mycobacterium tuberculosis has been resolved in the closed state. Numerous experiments have attempted to trap the channel in its open state but they did not succeed in obtaining a structure. A gating mechanism has been proposed based on different experimental data but there is no experimental technique available to follow this process in atomic details. In addition, it has been shown that a decrease of the lipid bilayer thickness lowered MscL activation energy and stabilized a structurally distinct closed channel intermediate. Here, we use atomistic molecular dynamics simulations to investigate the effect of the lipid bilayer thinning on our model of the structure of the Escherichia coli. We thoroughly analyze simulations of the channel embedded in two pre-equilibrated membranes differing by their hydrophobic tail length (DMPE and POPE). The MscL structure remains stable in POPE, whereas a distinct structural state is obtained in DMPE in response to hydrophobic mismatch. This latter is obtained by tilts and kinks of the transmembrane helices, leading to a widening and a diminution of the channel height. Part of these motions is guided by a competition between solvent and lipids for the interaction with the periplasmic loops. We finally conduct a principal component analysis of the simulation and compare anharmonic motions with harmonic ones, previously obtained from a coarse-grained normal mode analysis performed on the same structural model. Significant similarities exist between low-frequency harmonic motions and those observed with essential dynamics in DMPE. In summary, change in membrane thickness permits to accelerate the conformational changes involved in the mechanics of the E. coli channel, providing a closed structural intermediate en route to the open state. These results give clues for better understanding why the channel activation energy is lowered in a thinner membrane.