John R. Liggins

Energetics of the specific binding interaction of the first three zinc fingers of the transcription factor TFIIIA with its cognate DNA sequence.

The energetics of the specific interaction of a protein fragment (zf1‐3) containing the three N‐terminal zinc fingers of the Xenopus laevis transcription factor TFIIIA with its cognate DNA sequence, contained in a 15 bp DNA duplex were studied using isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and fluorescence titration. The use of both ITC and DSC is necessary to provide values for the thermodynamic parameters that have been corrected for thermal fluctuations of the interacting molecules. In the temperature range from 13°C to 45°C (where all the binding reaction components are folded), formation of the complex is enthalpically driven with a negative heat capacity effect (ΔCp). In this respect, the binding reaction of zf1‐3 is similar to those of other proteins that bind in the major groove of DNA. It is dissimilar to the association reactions of proteins, however, that bind in the minor groove of DNA and that are driven by a dominating entropy factor. Comparison of the experimental values of ΔHass and ΔCp with expected values of these parameters, calculated from the burial of polar and nonpolar molecular surfaces, indicates that the polar groups at the protein/DNA interface are not completely dehydrated upon formation of the complex. It also seems that the expected large positive entropy of dehydration upon forming the zfl‐3/DNA complex (∼1900 J • K−1 • mol−1) cannot be balanced by the reduction in translational/rotational and configurational freedom of the protein to the level of the observed entropy of binding (38 J • K−1 • mol−1). It is suggested that the additional negative entropy contribution comes from a damping of torsional motions in the DNA duplex. Proteins 2000;41:50–62.

Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work

Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg2+, to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.

The importance of protein-protein interactions for optimising oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin--manganese stabilising protein.

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein–protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.

Entropy and biological systems: Experimentally-investigated entropy-driven stacking of plant photosynthetic membranes

According to the Second Law of Thermodynamics, an overall increase of entropy contributes to the driving force for any physicochemical process, but entropy has seldom been investigated in biological systems. Here, for the first time, we apply Isothermal Titration Calorimetry (ITC) to investigate the Mg2+-induced spontaneous stacking of photosynthetic membranes isolated from spinach leaves. After subtracting a large endothermic interaction of MgCl2 with membranes, unrelated to stacking, we demonstrate that the enthalpy change (heat change at constant pressure) is zero or marginally positive or negative. This first direct experimental evidence strongly suggests that an entropy increase significantly drives membrane stacking in this ordered biological structure. Possible mechanisms for the entropy increase include: (i) the attraction between discrete oppositely-charged areas, releasing counterions; (ii) the release of loosely-bound water molecules from the inter-membrane gap; (iii) the increased orientational freedom of previously-aligned water dipoles; and (iv) the lateral rearrangement of membrane components.

Putative Functional Role for the Invariant Aspartate 263 Residue of Rhodospirillum rubrum Rubisco

Although aspartate residue D263 of Rhodospirillum rubrum Rubisco is close to the active site and invariant in all reported Rubiscos, its possible functional and structural roles in Rubisco activity have not been investigated. We have mutagenised D263 to several selected amino acids (asparagine, alanine, serine, glutamate, and glutamine) to probe possible roles in facilitating proton movements within the active site and maintaining structural positioning of key active-site groups. The mutants have been characterized by kinetic methods and by differential scanning calorimetry (DSC) to examine the effects of the substitutions on the stability of the folded state. We show that D263 is essential for maintaining effective levels of catalysis with the mutations reducing carboxylation variously by up to 100-fold but having less than 10% effect on the carboxylase/oxygenase specificity of the catalytic reaction. Removing the charge of the residue 263 side chain significantly strengthens binding of the activating (carbamylating) CO(2) molecule. In contrast, a charge on the 263 site has only a small influence on binding of the positively charged Mg(2+) ion, suggesting that the local protein structure provides different shielding of the formal charges on the Mg(2+) ion and the epsilon-lysine group of K191. Interestingly, introduction of an internal cavity (D263S and D263A) and insertion of an extra -CH(2)- group (D263E and D263Q) have opposite effects on catalysis, the former relatively small and the latter much larger, suggesting that the extra side-chain group induces a specific structural distortion that inhibits formation of the transition state. As the DSC results show that the mutations only slightly increase the kinetic stability of the folded state, we conclude that the rate-limiting (activated) step of unfolding involves substantial unfolding of the structure but not in the region of site 263. In summary, interaction of D263 with H287 of a largely electrostatic nature appears critical for maintaining correct positioning of catalytic groups in the active site. The conservation of D263 can thus be accounted for by its contribution to the maintenance of a finely tuned structure in this region abutting the active site.