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Conformational protein dynamics isknown to be hampered in amorphous matrixes upon dehydration, both in the absence and in the presenceof glass forming disaccharides, like trehalose, resulting in enhancedprotein thermal stability. To shed light on such matrix effects, wehave compared the retardation of protein dynamics in photosyntheticbacterial reaction centers (RC) dehydrated at controlled relativehumidity in the absence (RC films) or in the presence of trehalose(RC–trehalose glasses). Small scale RC dynamics, associatedwith the relaxation from the dark-adapted to the light-adapted conformation,have been probed up to the second time scale by analyzing the kineticsof electron transfer from the photoreduced quinone acceptor (QA–) to the photoxidized primary donor (P+) as a function of the duration of photoexcitation from 7ns (laser pulse) to 20 s. A more severe inhibition of dynamics isfound in RC–trehalose glasses than in RC films: only in thelatter system does a complete relaxation to the light-adapted conformationoccur even at extreme dehydration, although strongly retarded. Togain insight into the large scale RC dynamics up to the time scaleof days, the kinetics of thermal denaturation have been studied at44 °C by spectral analysis of the Qxand Qybands of the RC bacteriochlorincofactors, as a function of the sugar/protein molar ratio, m, varied between 0 and 104. Upon increasing m, denaturation is slowed progressively, and above m∼ 500 the RC is stable at least for several days.The stronger retardation of RC relaxation and dynamics induced bytrehalose is discussed in the light of a recent molecular dynamicssimulation study performed in matrixes of the model protein lysozymewith and without trehalose. We suggest that the efficiency of trehalosein retarding RC dynamics and preventing thermal denaturation stemsmainly from its propensity to form and stabilize extended networksof hydrogen bonds involving sugar, residual water, and surface residuesof the RC complex and from its ability of reducing the free volumefraction of protein alone matrixes. [ABSTRACT FROM AUTHOR]

The coupling between electron transfer (ET) and the conformational dynamics of the cofactor−protein complex in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroidesin water/glycerol solutions or embedded in dehydrated poly(vinyl alcohol) (PVA) films or trehalose glasses is reported. Matrix effects were studied by time-resolved 95 GHz high-field electron paramagnetic resonance (EPR) spectroscopy at room (290 K) and low (150 K) temperature. ET from the photoreduced quinone acceptor (QA•−) to the photo-oxidized donor (P865•) is strongly matrix-dependent at room temperature: In the trehalose glasses, the recombination kinetics of P865••−, probed by EPR and optical spectroscopies, is faster and broadly distributed as compared to that of RCs in solution, reflecting the inhibition of the RC relaxation from the dark- to the light-adapted conformational substate and the hindrance of substate interconversion. Similarly accelerated kinetics was observed also in PVA at a water-to-RC molar ratio 10-fold lower than in trehalose. Despite the matrix dependence of the ET kinetics, continuous-wave (cw) EPR and electron spin echo (ESE) analyses of the photogenerated P865•橷 QA•−radical ions and P865••−radical pairs do not reveal significant matrix effects, at either 290 or 150 K, indicating no change in the molecular radical-pair configuration of the P865•橷 QA•−cofactors. Furthermore, the field dependences of the transverse relaxation times T2of QA•−essentially coincide in trehalose and PVA at 290 K. T2is similar in these two matrixes and in the glycerol/water system at 150 K, implying that the librational dynamics of QA•−are also unaffected by the matrix. We infer that the relative geometry of the primary donor and acceptor, as well as the local dynamics and hydrogen bonding of QAin its binding pocket, are not involved in the stabilization of P865••−. We suggest that the RC relaxation occurs rather by changes throughout the protein/solvent system. The control of the RC dynamics and ET by the environment is discussed, particularly with respect to the extraordinary efficacy of trehalose matrixes in restricting the RC motional degrees of freedom at elevated temperatures. [ABSTRACT FROM AUTHOR]

The coupling between electron transfer and protein dynamics has been investigated in reaction centers (RCs) from the wild type (wt) and the carotenoid-less strain R26 of the photosynthetic bacterium Rhodobacter sphaeroides. Recombination kinetics between the primary photoreduced quinone acceptor (QA−) and photoxidized donor (P+) have been analyzed at room temperature in RCs incorporated into glassy trehalose matrices of different water/sugar ratios. As previously found in R26 RCs, also in the wt RC, upon matrix dehydration, P+QA−recombination accelerates and becomes broadly distributed, reflecting the inhibition of protein relaxation from the dark-adaptedto the light-adaptedconformation and the hindrance of interconversion between conformational substates. While in wet trehalose matrices (down to ∼one water per trehalose molecule) P+QA−recombination kinetics are essentially coincident in wt and R26 RCs, more extensive dehydration leads to two-times faster and more distributed kinetics in the carotenoid-containing RC, indicating a stronger inhibition of the internal protein dynamics in the wt RC. Coarse-grained Brownian dynamics simulations performed on the two RC structures reveal a markedly larger flexibility of the R26 RC, showing that a rigid core of residues, close to the quinone acceptors, is specifically softened in the absence of the carotenoid. These experimental and computational results concur to indicate that removal of the carotenoid molecule has long-range effects on protein dynamics and that the structural/dynamical coupling between the protein and the glassy matrix depends strongly upon the local mechanical properties of the protein interior. The data also suggest that the conformational change stabilizing P+QA−is localized around the QAbinding pocket. [ABSTRACT FROM AUTHOR]