No abstract

The spectroscopic analysis of large biomolecules is important in applications such as biomedical diagnostics and pathogen detection 1,2 , and spectroscopic techniques can detect such molecules at the nanogram level or lower. However, spectroscopic techniques have not been able to probe the structure of large biomolecules with similar levels of sensitivity. Here we show that superchiral electromagnetic fields 3 , generated by the optical excitation of plasmonic planar chiral metamaterials 4,5 , are highly sensitive probes of chiral supramolecular structure. The differences in the effective refractive indices of chiral samples exposed to left-and right-handed superchiral fields are found to be up to 10 6 times greater than that those observed in optical polarimetry measurements, thus allowing picogram quantities of adsorbed molecules to be characterised. The largest differences are observed for biomolecules that possess chiral planar 2 sheets, such as proteins with high β-sheet content, which suggest that this approach could form the basis for assaying technologies capable of detecting amyloid diseases and certain types of viruses.Since the building blocks of life are chiral molecular units such as amino acids and sugars, biomacromolecules formed from these units also exhibit chirality on molecular and supramolecular scales. Chirally sensitive (chiroptical) spectroscopic techniques, such as circular dichroism (CD), optical rotatory dispersion (ORD) and Raman optical activity (ROA), are therefore especially incisive probes of the threedimensional aspects of biomacromolecular structure and are widely used in biomolecular science 1,2 . Chiroptical methods typically measure small differences, or dissymmetries, in the interaction of left-and right-circularly polarised light, the chiral probe, with a chiral material 2 . However, the inherent weakness of these existing chiroptical phenomena usually restricts their application to samples of microgram level. Recently, Tang and Cohen 3 postulated that under certain circumstances superchiral electromagnetic fields could be produced that display greater chiral asymmetry than circularly polarised plane light waves. We have realised such superchiral electromagnetic fields are generated in the near fields of PCMs, and can greatly enhanced the sensitivity of a chiroptical measurement, enabling us to detect and characterise just a few picograms of a chiral material.PCMs were first fabricated, and shown to display large chiroptical effects such as optical rotation, by Schwanecke and co-workers 4 and Gonokami and co-workers 5 .The PCMs used in this study, Fig. 1 (a), are composed of left or right handed (LH / RH) Au gammadions, of length 400 nm and thickness 100 nm (plus a 5 nm Cr adhesion layer) deposited on a glass substrate and arranged in a square lattice with a periodicity of 800 nm. As a control we repeated all experiments using a metamaterial composed of achiral crosses with the same thickness and periodicity as the gammadions: these structures showed no dissymmetry in excita...

Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as α-helix and β-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising "molten globule" intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.

The recently identified plant photoreceptor UVR8 triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light via an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine re-tunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs.