Retardation of Protein Dynamics by Trehalose in Dehydrated Systems of Photosynthetic Reaction Centers. Insights from Electron Transfer and Thermal Denaturation Kinetics

Abstract

Conformational protein dynamics is known to be hampered in amorphous matrixes upon dehydration, both in the absence and in the presence of glass forming disaccharides, like trehalose, resulting in enhanced protein thermal stability. To shed light on such matrix effects, we have compared the retardation of protein dynamics in photosynthetic bacterial reaction centers (RC) dehydrated at controlled relative humidity in the absence (RC films) or in the presence of trehalose (RC-trehalose glasses). Small scale RC dynamics, associated with the relaxation from the dark-adapted to the light-adapted conformation, have been probed up to the second time scale by analyzing the kinetics of electron transfer from the photoreduced quinone acceptor (QA(-)) to the photoxidized primary donor (P(+)) as a function of the duration of photoexcitation from 7 ns (laser pulse) to 20 s. A more severe inhibition of dynamics is found in RC-trehalose glasses than in RC films: only in the latter system does a complete relaxation to the light-adapted conformation occur even at extreme dehydration, although strongly retarded. To gain insight into the large scale RC dynamics up to the time scale of days, the kinetics of thermal denaturation have been studied at 44 °C by spectral analysis of the Qx and Qy bands of the RC bacteriochlorin cofactors, as a function of the sugar/protein molar ratio, m, varied between 0 and 10(4). 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 by trehalose is discussed in the light of a recent molecular dynamics simulation study performed in matrixes of the model protein lysozyme with and without trehalose. We suggest that the efficiency of trehalose in retarding RC dynamics and preventing thermal denaturation stems mainly from its propensity to form and stabilize extended networks of hydrogen bonds involving sugar, residual water, and surface residues of the RC complex and from its ability of reducing the free volume fraction of protein alone matrixes.

Local water sensing: water exchange in bacterial photosynthetic reaction centers embedded in a trehalose glass studied using multiresonance EPR

“…Upon progressive dehydration of the trehalose matrix, the kinetics of P þ 865 Q À A charge recombination becomes faster and exhibits widely distributed rate constants. 23,28,54,56 Thus, it mimics at room temperature the bRC recombination kinetics observed at cryogenic temperatures in the water-glycerol system when frozen in the dark. 57,58 A reasonable explanation for this matrix effect is the stronger dynamic coupling between sugar and protein via hydrogenbonding networks in a dehydrated trehalose matrix.…”

“…The resulting sugar matrix has very different properties: at low humidity and the corresponding low hydration level the protein-matrix system is rigid with only 0.5 water molecules per sugar molecule; at higher humidity and hydration level the sugar matrix is less rigid and contains more water molecules as potential partners for hydrogen bonding to the protein and to the sugar matrix. Different hydration levels, therefore, affect the protein dynamics differently: at r = 11%, the dynamics is blocked, 54,59,60 at least on the timescale of P þ 865 ÀQ À A charge recombination, but is not affected at r = 74%. Such an effect of the dry trehalose matrix, to accomplish inhibition of internal protein dynamics and, thereby, leading to bio-protection of proteins against thermal denaturation, reflects a strong ''slaving'' of the protein dynamics to that of the embedding matrix.…”

“…53 The effect of bRC coatings on a trehalose glass matrix has been thoroughly studied previously using laser-flash optical and EPR spectroscopies. 23,28,30,32,54 In bRCs, the coating with trehalose affects the electron-transfer step between the primary and secondary quinone acceptors 32 which is a conformationally gated process. 55 The trehalose matrix also influences the lifetime of the chargeseparated radical-pair state, P þ 865 Q À A .…”

“…It blocks the fluctuations between conformational substates of the bRC, 28 as put forward in the ''anchorage hypothesis''. 31 Two relative hydration levels, r, were chosen in this work that result in a different extent of slaving the protein dynamics to the embedding sugar matrix: (i) r = 11% for which the protein dynamics is arrested on the time scale of seconds, 54 and (ii) r = 74% for which the dynamics is only mildly retarded as compared to solution. 59 Using EDNMR we aim to probe directly if the dehydration of the trehalose matrix results in a changed hydration level of the inner protein core surrounding the native cofactors, P þ 865 and Q À A , and of the protein surface at the labeled site of cysteine 156.…”

Interaction of Single Cells with 2D Organic Monolayers: A Scanning Electrochemical Microscopy Study

“…Decomposition of frequency distributions of # into four Gaussian components were performed by least-squared minimization routines based on grid search algorithm [31] using in-house developed software. [32] Frequency distributions of #, F(#), were fitted to Equation (3):…”