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 Mg(2+)-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.

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 Mg(2+), 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.

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.

Conformation of prion protein repeat peptides probed by FRET measurements and molecular dynamics simulations.

We report the combined use of steady-state fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to investigate conformational distributions of the prion protein (PrP) repeat system. FRET was used for the first time to probe the distance, as a function of temperature and pH, between a donor Trp residue and an acceptor dansyl group attached to the N-terminus in seven model peptides containing one to three repeats of the second decarepeat of PrP from marsupial possum (PHPGGSNWGQ)nG, and one and two human PrP consensus octarepeats (PHGGGWGQ)nG. In multirepeat peptides, single-Trp mutants were made by replacing other Trp(s) with Phe. As previous work has shown PrP repeats do not adopt a single preferred stable conformation, the FRET values are averages reflecting heterogeneity in the donor-acceptor distances. The T-dependence of the conformational distributions, and derived average dansyl-Trp distances, were obtained directly from MD simulation of the marsupial dansyl-PHPGGSNWGQG peptide. The results show excellent agreement between the FRET and MD T-dependent distances, and demonstrate the remarkable sensitivity and reproducibility of the FRET method in this first-time use for a set of disordered peptides. Based on the results, we propose a model involving cation-pi or pi-pi His-Trp interactions to explain the T- (5-85 degrees C) and pH- (6.0, 7.2) dependencies on distance, with HW i, i + 4 or WH i, i + 4 separations in sequence being more stable than HW i, i + 6 or WH i, i + 6 separations. The model has peptides adopting loosely folded conformations, with dansyl-Trp distances very much less than estimates for fully extended conformations, for example, approximately 16 vs. 33, approximately 21 vs. 69, and approximately 22 vs. 106 A for 1-3 decarepeats, and approximately 14 vs. 25 and approximately 19 vs. 54 A for 1-2 octarepeats, respectively. The study demonstrates the usefulness of combining FRET with MD, a combination reported only once previously. Initial "mapping" of the conformational distribution of flexible peptides by simulation can assist in designing and interpreting experiments using steady-state intensity methods, and indicating how time-resolved or anisotropy methods might be used.

The energetics of specific binding of AT-hooks from HMGA1 to target DNA.

The interaction of the second and third AT-hooks of HMGA1 (formerly HMGI/Y), which bind selectively in the minor groove of an AT-rich DNA sequence, was studied at different temperatures and ionic strengths by spectropolarimetry, spectrofluorimetry, isothermal titration calorimetry and differential scanning calorimetry. The data show that binding of the ten amino acid core element of the two AT-hooks, which penetrates deep into the minor groove, is entropically driven: both the entropy and enthalpy of association of the peptides to the target DNA are positive up to 50 degrees C. The seven amino acid extension of the core in the second AT-hook, which extends out from the minor groove and loops over the phosphodiester backbone, adds a substantial negative enthalpic component into the binding of the 17 residue DBD2 peptide to DNA that corresponds in magnitude to the enthalpy of formation of two hydrogen bonds. The ionic strength dependence of the association constant allowed an estimation of the electrostatic component of binding and, by subtraction, the contribution of the non-electrostatic component, which results from dehydration of the contacting surfaces and makes up almost 70% of the total energy of complex formation. The exceptionally large positive entropy and enthalpy of association of the core AT-hook peptides with target DNA suggest that the water, which is removed from the minor groove of DNA upon binding, is in a highly ordered state. Acetylation of the lysine residue in the second AT-hook, which corresponds to Lys65 of HMGA1, has little effect on the DNA binding; so it appears that repression of the hIFNbeta gene, which follows this modification, is not a direct result of the abrogation of DNA binding.