Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Rational identification and characterisation of peptide ligands for targeting polysialic acid.

The alpha-2,8-linked form of the polysaccharide polysialic acid (PSA) has widespread implications in physiological and pathological processes, ranging from neurological development to disease progression. Though the high electronegativity and excluded volume of PSA often promotes interference of biomolecular interactions, PSA-binding ligands have important implications for both biological processes and biotechnological applications. As such, the design, identification, and characterisation of novel ligands towards PSA is critical for expanding knowledge of PSA interactions and achieving selective glycan targeting. Here, we report on a rational approach for the identification of alpha-2,8-PSA-binding peptides, involving design from the endogenous ligand Siglec-11 and multi-platform characterisation of peptide binding. Microarray-based examination of peptides revealed charge and sequence characteristics influencing peptide affinity to PSA, and carbohydrate-peptide binding was further quantified with a novel fluorescence anisotropy assay. PSA-binding peptides exhibited specific binding to polymeric SA, as well as different degrees of selective binding in various conditions, including competition with PSA of alternating 2,8/9-linkages and screening with PSA-expressing cells. A computational study of Siglec-11 and Siglec-11-derived peptides offered synergistic insight into ligand binding. These results demonstrate the potential of PSA-binding peptides for selective targeting and highlight the importance of the approaches described herein for the study of carbohydrate interactions.

Microarrays for the screening and identification of carbohydrate-binding peptides.

The development of carbohydrate-binding ligands is crucial for expanding knowledge on the glycocode and for achieving systematic carbohydrate targeting. Amongst such ligands, carbohydrate-binding peptides (CBPs) are attractive for use in bioanalytical and biomedical systems due to their biochemical and physicochemical properties; moreover, given the biological significance of lectin-carbohydrate interactions, these ligands offer an opportunity to study peptide sequence and binding characteristics to inform on natural target/ligand interactions. Here, a high-throughput microarray screening technique is described for the identification and study of CBPs, with a focus on polysialic acid (PSA), a polysaccharide found on neural stem cells. The chemical and biological uniqueness of PSA suggests that an ability to exclusively target this glycan may promote a number of diagnostic and therapeutic applications. PSA-binding peptides from phage display screening and from epitope mapping of an scFv for oligosialic acid were screened in an optimized microarray format with three ligand density conditions. Hypothesis-driven mutations were additionally applied to select peptides to modulate peptide affinity and selectivity to PSA. Peptide compositional and positional analyses revealed the significance of various residues for PSA binding and suggested the importance of basic residue positioning for PSA recognition. Furthermore, selectivity studies performed directly on microarrays with chondroitin sulfate A (CS-A) demonstrated the value of screening for both affinity and selectivity in the development of CBPs. Thus, the integrated approach described, with attention to design strategy, screening, and peptide characterization, successfully identified novel PSA-binding ligands and offers a platform for the identification and study of additional polysaccharide-binding peptides.