Expected and observed effects of volume exclusion on the free energy of rigid and flexible macromolecules in crowded and confined systems, and consequent effects of crowding and confinement on macromolecular reaction rates and equilibria are summarized. Findings from relevant theoretical/simulation and experimental literature published from 2004 onward are reviewed. Additional complexity arising from the heterogeneity of local environments in biological media, and the presence of nonspecific interactions between macromolecules over and above steric repulsion are discussed. Theoretical and experimental approaches to the characterization of crowding-and confinement-induced effects in systems approaching the complexity of living organisms are suggested.

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.protein misfolding | amyloid aggregation | toxic oligomer | cryoelectron microscopy | neurodegeneration

The conformational properties of a 16 residue peptide, corresponding to the second beta-hairpin of the B1 domain of protein G, have been studied by nuclear magnetic resonance spectroscopy (NMR). This fragment is monomeric under our experimental conditions and in pure water adopts a population containing up to 40% native-like beta-hairpin structure. The detection by NMR of a native-like beta-hairpin in aqueous solution, reported here for the first time, indicates that these structural elements may have an important role in the early steps of protein folding. It also provides a good model to study in detail the sequence determinants of beta-hairpin structure stability, as has been done with alpha-helices.

Biochemical processes take place in heterogeneous and highly volume occupied or crowded environments that can considerably influence the reactivity and distribution of participating macromolecules. Here we summarize the thermodynamic consequences of excluded volume and longer-ranged nonspecific intermolecular interactions for macromolecular reactions in volume-occupied media. In addition, we summarize and compare the information content of studies of crowding in vitro and in vivo. We emphasize the importance of characterizing the behavior not only of labeled tracer macromolecules, but also the composition and behavior of unlabeled macromolecules in the immediate vicinity of the tracer. Finally, we propose strategies for extending quantitative analyses of crowding in simple model systems to increasingly complex media up to and including intact cells.