203 Polymers through pores: single-molecule experiments with nucleic acids,polypeptides and polysaccharides

The translocation of polymers through pores has been examined for almost two decades with an emphasis on nucleic acids. There are also interesting circumstances in biology where polypeptides and polysaccharides pass through transmembrane pores, and our laboratory has been investigating examples of them. Single-molecule nucleic acid sequencing by nanopore technology is an emerging approach for ultrarapid genomics. Strand sequencing with engineered protein nanopores is a viable technology which has required advances in four areas: nucleic acid threading, nucleobase identification, controlled strand translocation, and nanopore arrays (Bayley, 2012). The latter remain a pressing need and our attempts to improve arrays will be described. In several physiological situations, folded proteins pass through transmembrane pores. We have developed a model system comprising mutant thioredoxins as the translocated proteins, and staphylococcal alpha-hemolysin, as the pore. Our findings support a mechanism in which there is local unfolding near the terminus of the polypeptide that enters the pore. The remainder of the protein then unfolds spontaneously and diffuses through the pore into the recipient compartment (Rodriguez-Larrea & Bayley, 2013). We have also examined the pore formed by the E. coli outer membrane protein Wza, which transports capsular polysaccharide from its site of synthesis to the outside of the cell. We made mutant open forms of the pore and screened blockers for them by electrical recording in planar bilayers. The most effective blocker binds in the alpha-helix barrel of Wza, a site accessible from the external medium, and therefore, a prospective target for antibiotics (Kong et al., 2013).     

187 Plucking the high hanging fruit: a systematic approach for targeting protein interfaces

Development of specific ligands for protein targets that help decode the complexities of protein–protein interaction networks is a key goal for the field of chemical biology. Despite the emergence of powerful in silico and experimental high-throughput screening strategies, the discovery of synthetic ligands that selectively modulate protein–protein interactions remains a challenge for the chemical biologists. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these domains, we can modulate the function of a particular protein with metabolically-stable synthetic molecules (Raj et al., 2013). This presentation will discuss computational approaches (Bullock et al., 2011; Jochim & Arora, 2010) to identify targetable interfaces along with synthetic methods (Patgiri et al., 2008; Tosovska & Arora, 2010) to develop protein domain mimics (PDMs) as modulators of intracellular protein–protein interactions (Henchey et al., 2010; Patgiri et al., 2011).     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.