Session L43: Invited Session: Precursors to the Folding and Aggregation of Biological Molecules

Postranslational modifications significantly alter the binding-folding pathways of proteins associating with DNA

Many important regulators of gene activity are natively disordered, but fully or partially order when they bind to their targets on DNA. Interestingly, the ensembles of disordered states for such free proteins are not structurally featureless, but can qualitatively differ from protein to protein. In particular, in random coil like states the chains are swollen, making relatively few contacts, while in molten globule like states a significant collapse occurs, with ensuing high density of intra-protein interactions. Furthermore, since many DNA binding proteins are positively charged polyelectrolytes, the electrostatic self-repulsion also influences the degree of collapse of the chain and its conformational preferences in the free state and upon binding to DNA. In our work, we have found that the nature of the natively disordered ensemble significantly affects the way the protein folds upon binding to DNA. In particular, we showed that posttranslational modifications of amino acid residues, such as lysine acetylation, can alter the degree of collapse and conformational preferences for a free protein, and also profoundly impact the binding affinity and pathways for the protein DNA association. These trends will be discussed in the context of DNA interacting with various histone tails and the p53 protein.   

Mesoscopic Aggregation in Protein Solutions

Long-lived mesoscopic clusters of a dense protein liquid are observed in concentrated solutions of numerous proteins. These clusters are a necessary kinetic intermediate for the formation of solid aggregates of native and misfolded protein molecules. We propose a novel physicochemical mechanism, by which the clusters consist of an off-equilibrium mixture of single protein molecules and long-lived protein-containing complexes. The puzzling mesoscopic size of the clusters is determined by the lifetime of the complexes and their diffusivity. We have predicted and observed a number of interesting kinetic and thermodynamic behaviors that are associated with the mesoscopic clusters. These behaviors include: (a) Ostwald-like ripening of the clusters (b) a crossover to long-range density fluctuations at high concentrations; (c) a universal, diffusion-like scaling of the autocorrelation function of light scattered off the protein solution; (d) non-trivial dependencies of the cluster size and volume fraction on the protein concentration in the solution. Our analysis of the anisotropic Coulomb interactions suggests for mesoscopic clusters to form in lysozyme solutions, protein molecules must undergo conformational changes.   

The impact of conformational transformations on the pathways and kinetics of protein self-assembly into extended matrices

The concept of a folding funnel with kinetic traps is used to describe folding of individual proteins. Using in situ AFM to investigate both collagen fibril and S-layer membrane assembly on mica, we show this concept is equally valid during self-assembly of proteins into extended matrices. Moreover, considering the conformational changes required to achieve the ordered structure is critical to understanding the pathway to the final state and the kinetics of assembly. In the collagen system, both the pathway and the final conformational state can be finely tuned by varying the ionic strength. This alters the relative strengths of the collagen-collagen and collagen-mica binding free energy, which we quantify using dynamic force spectroscopy. Moreover, when conditions result in the ordered D-band structure of collagen, the emergence of order catalyzes the further transformation leading to non-linear attachment kinetics. In the S-layer system, there is a kinetic trap associated with conformational differences between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of an amorphous precursor phase, however, they then track along two different pathways. One leads directly to the final low-energy state and the other to the kinetic trap. Over time, the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation we find the energy barriers to formation of either state to be nearly identical, but once the high-energy state forms, the barrier to transformation to the low-energy state is large. Thus the transient state exhibits the characteristics of a kinetic trap in a folding funnel.   

The role of proteins and peptides in shaping the structure and microstructure of biogenic and biomimetic crystals

In the course of biomineralization organisms form crystals which demonstrate superior characteristics as compared to their non-biogenic counterparts. One of the main reasons for this is that these crystals are actually hybrid nano-composites. In fact, each biogenic single crystal is not only encapsulated by an intercrystalline organic phase but in addition there are intracrystalline proteins within each individual single crystal. These proteins are the key players in the formation of biominerals and are vital in the precursor phase of their formation. I will show that both in biogenic and biomimetic crystals, these proteins have a major effect on their atomic structure and microstructure. This effect is in the form of lattice distortions which relax upon mild annealing. The distortions can be detected by means of high-resolution synchrotron diffraction. The activation energy of the relaxation of the lattice distortions is rather low and is comparable to the energy needed for protein unfolding. Furthermore, it will be shown that these intracrystalline proteins can stabilize metastable phases of calcium carbonate and even stabilize a previously unidentified twin law in calcite.