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The ability to determine the identity of specific proteins is a critical challenge in many areas of cellular and molecular biology, and in medical diagnostics. Here, we present a macine learning aided microfluidic protein characterisation strategy that within a few minutes generates a three-dimensional fingerprint of a protein sample indicative of its amino acid composition and size and, thereby, creates a unique signature for the protein. By acquiring such multidimensional fingerprints for a set of ten proteins and using machine learning approaches to classify the fingerprints, we demonstrate that this strategy allows proteins to be classified at a high accuracy, even though classification using a single dimension is not possible. Moreover, we show that the acquired fingerprints correlate with the amino acid content of the samples, which makes it is possible to identify proteins directly from their sequence without requiring any prior knowledge about the fingerprints. These findings suggest that such a multidimensional profiling strategy can lead to the development of a novel method for protein identification in a microfluidic format., Protein classification and identification from their multidimensional fingerprints obtained on a microfluidic chip.

Neurodegenerative diseases such as Alzheimer's and Parkinson's are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging., Introducing ThX, a next-generation ThT derivative that allows for the early detection of amyloid aggregates at the bulk and single-aggregate levels.

Investigations of protein folding, unfolding and stability are critical for the understanding of the molecular basis of biological structure and function. We describe here a microfluidic approach to probe the unfolding of unlabelled protein molecules in microliter volumes. We achieve this objective through the use of a microfluidic platform, which allows the changes in molecular diffusivity upon folding and unfolding to be detected directly. We illustrate this approach by monitoring the unfolding of bovine serum albumin in solution as a function of pH. These results show the viability of probing protein stability on chip in small volumes.

Parkinson's disease is one of the major neurodegenerative disorders affecting the ageing populations of the modern world. One of the hallmarks of this disease is the deposition of aggregates, mainly of the small pre-synaptic protein α-synuclein (AS), in the brains of patients. Several very significantly modified forms of AS have been found in these deposits including those resulting from truncations of the protein at its C-terminus. Here, we report how two physiologically relevant C-terminal truncations of AS, AS(1-119) and AS(1-103), where either half or virtually all of the C-terminal domain, respectively, has been truncated, affect the mechanism of AS aggregation and the properties of the fibrils formed. In particular, we have found that the deletion of these C-terminal residues induces a shift of the pH region where autocatalytic secondary processes dominate the kinetics of AS aggregation towards higher pH values, from AS wild-type (pH 3.6-5.6) to AS(1-119) (pH 4.2-7.0) and AS(1-103) (pH 5.6-8.0). In addition, we found that both truncated variants formed protofibrils in the presence of lipid vesicles, but only those formed by AS(1-103) had the capacity to convert readily into mature fibrils. These results suggest that electrostatics play an important role in secondary nucleation, a key factor in aggregate proliferation, and in the conversion of AS fibrils from protofibrils to mature fibrils. In particular, our results demonstrate that sequence truncations of AS can shift the pH range where autocatalytic proliferation of fibrils is possible into the neutral, physiological regime, thus providing an explanation of the increased propensity of the C-truncated variants to aggregate in vivo.

The aggregation of the amyloid β peptide (Aβ42), which is linked to Alzheimer's disease, can be altered significantly by modulations of the peptide's intermolecular electrostatic interactions. Variations in sequence and solution conditions have been found to lead to highly variable aggregation behaviour. Here we modulate systematically the electrostatic interactions governing the aggregation kinetics by varying the ionic strength of the solution. We find that changes in the solution ionic strength induce a switch in the reaction pathway, altering the dominant mechanisms of aggregate multiplication. This strategy thereby allows us to continuously sample a large space of different reaction mechanisms and develop a minimal reaction network that unifies the experimental kinetics under a wide range of different conditions. More generally, this universal reaction network connects previously separate systems, such as charge mutants of the Aβ42 peptide, on a continuous mechanistic landscape, providing a unified picture of the aggregation mechanism of Aβ42.

Resonances from two distinct thiazolidine ring conformers have been resolved in the 13C CP-MAS spectra of frozen aqueous solutions of penicillins; the relative intensities of the resonances correlate well with previous estimates of the populations of the different conformers in liquid aqueous solutions.

Protein homeostasis refers to the ability of cells to generate and regulate the levels of their constituent proteins in terms of conformations, interactions, concentrations and cellular localisation. We discuss here an approach in which physico-chemical properties of proteins and their environments are used to understand the underlying principles governing this process, which is crucial in all living systems. By adopting the strategy of characterising the origins of specific diseases to inform us about normal biology, we are bringing together methods and concepts from chemistry, physics, engineering, genetics and medicine. In particular, we are using a combination of in vitro, in silico and in vivo approaches to study protein homeostasis through the analysis of the effects that result from its perturbation in a select group of specific proteins, from either amino acid mutations, or changes in concentration and solubility, or interactions with other molecules. By developing a coherent and quantitative description of such phenomena, we are finding that it is possible to shed new light on how the physical and chemical properties of the cellular components can provide an understanding of the normal and aberrant behaviour of living systems. Through such an approach it is possible to provide new insights into the origin and consequences of the failure to maintain homeostasis that is associated with neurodegenerative diseases, in particular, and the phenomenon of ageing, in general, and hence provide a framework for the rational design of therapeutic approaches.

Comparison of experimental and calculated 23Na NMR spectra of complexes containing lanthanide cations indicates that paramagnetic effects in MAS NMR can yield quantitative structural information.

A substantial improvement in the resolution of 13C MAS NMR spectra of paramagnetic lanthanide acetates results from replacement of the protons by deuterons; this is attributed to the removal, in the deuteriated sample, of residual broadening due to incomplete proton decoupling.

Complex formation between cytidine 5′-monophosphate (cmp) and lanthanoid(III) cations has been followed by n.m.r. spectroscopy, making use of induced shifts of the n.m.r. lines of cmp caused by the bound lanthanoid cations. Shift data have been obtained under a variety of conditions using most of the lanthanoids. These data show that the conformation of cmp is similar to that of adenosine 5′-monophosphate (amp). Relaxation studies employing GdIII as perturbing cation confirm the conclusions drawn from the shift data.

The use of lanthanoid complexes of ethylenediaminetetra-acetic acid, [M (edta)]–(M = La, Pr, Eu, and Gd), as n.m.r. shift and relaxation probes has enabled the determination of the conformations of adenosine 5′-monophosphate (amp) and cytidine 5′-monophosphate (cmp) at pH 7·5. The conformations are the same as those found for the molecules at pH 2·0 using lanthanoid aquo-ions as probes.

It is shown that the combination of double irradiation and a simple two-pulse spin echo sequence is a powerful and sensitive method for detecting homonuclear decoupling in a complex spectrum.

A series of ternary tin oxides has been studied by high-resolution solid state 119Sn magic angle spinning n.m.r. spectroscopy. Linear correlations were observed between the 119Sn chemical shift and the cation radius in compounds of the type MSnO3 and M2SnO4. Significant chemical shift anisotropies have been identified with bond-angle and bond-length distortions of the SnO6octahedra present in these phases, although no straightforward relationship was seen between the chemical shift tensor asymmetry and the crystallographic site symmetry. Spin–spin coupling was observed in several compounds between chemically equivalent but magnetically inequivalent 119Sn and 117Sn nuclei separated by Sn–O–Sn bonds.

A high-yield synthesis of [Ru3(µ-H)2(CO)9{µ3-PCH2CH2Si(OEt)3}](1) from [Ru3(CO)12](2) has been developed. This complex can be tethered to high surface area oxide supports. The alumina based material has been characterised by i.r. and u.v.–visible spectroscopies and 31P cross polarisation magic angle spinning n.m.r., all of which yield comparable data to that obtained for the precursor in solution. Comparison of the chemical shift anisotropy of (1)–alumina with that of a polycrystalline sample of [Ru3(µ-H)2(CO)9(µ3-PPh)](3) is consistent with (1) being firmly tethered to the oxide surface. Ruthenium K-edge extended X-ray absorption fine structure data were obtained for (1) and its alumina tethered version. Analysis of each required two Ru–Ru distances, as expected for the isosceles metal triangle in these complexes. For the tethered complex, these distance were 275(1) and 294(1) pm for the Ru–Ru and Ru–H–Ru units respectively.

High-resolution 31P n.m.r. spectra of a series of crystalline gold phosphine cluster compounds have been obtained using magic angle sample spinning. The spectra are interpreted on the basis of the known crystal structures, and are compared with corresponding spectra of the compounds in solution. With one exception, the clusters appear to be stereochemically rigid in the crystals at room temperature, although most are fluxional in solution, some even at low temperatures. In many cases separate resonances can be resolved in the solid-state spectra from the distinct phosphine environments of the cluster framework. A combination of solid-state and solution n.m.r. spectroscopy is likely to be extremely valuable in the characterisation of both the structure and dynamics of phosphine-containing cluster compounds.

Solid-state 29Si n.m.r. spectroscopy has been used to follow the kinetics of the hydration of tricalcium silicate and to provide details concerning the reaction course.

High-resolution solid-state 29Si n.m.r. and i.r. spectroscopies have been used to monitor the crystalline–amorphous transition of (Ca, Na)-and (Sr, Na)-exchanged zeolites A hydrothermally treated in the presence of densified krypton gas. Both techniques are capable of monitoring the transition on a smaller scale than X-ray diffraction and demonstrate that thermally stable gas fixation takes place in a truly amorphous glassy matrix.

A combination of lineshape analysis and magnetisation transfer experiments in variable temperature 13C cross-polarisation magic-angle-spinning n.m.r. spectroscopy has enabled the determination of rate and activation parameters for aromatic ring rotations over a motional frequency range of 10–2–106 Hz in crystalline penicillin derivatives.

The conformation of the amino-acid tryptophan in solution has been examined by n.m.r. methods. Coupling constants, nuclear Overhauser effects, and lanthanide perturbations were all analysed in terms of six conformers, each having one of three values for the Cα–Cβ dihedral angle (χ1) and one of two values for the Cβ–Cγ dihedral angle (χ2). A set of populations was obtained which was consistent, within reasonable limits, with all the n.m.r. data. It was found that one conformer, that having the side-chain trans to the carboxylic acid group and the ring perpendicular to the Cα–Cβ bond, was the dominant species in solution. There are similarities between the population distribution in solution and the distribution of dihedral angles found for tryptophan in protein crystal structures.

The conformations of ampicillin, penicillin G and penicillin V sulphoxide in aqueous solution have been studied by analysing the shift and relaxation perturbations to the nuclear magnetic resonance spectra induced by binding lanthanide cations. The conformations of the molecules in solution are very similar, despite differences observed in the solid state.

The effects on the n.m.r. spectrum of binding various lanthanide complexes to the title dicarboxylic acid in aqueous solution have been studied. It is shown using relaxation-time measurements that all lanthanides and complexes form similar adducts with the acid. The magnetic susceptibility tensor, which determines the shifts induced by a lanthanide, is not related simply to the geometry of the adduct. Rather, an averaged tensor is observed, whose symmetry is close to axial for lanthanides in the first half of the series, but often far from axial for those later in the series. It is proposed that this type of behaviour is general for lanthanide complexes in water.

2 H N.m.r. spectroscopy reveals the existence of two orientations of the cobaltocenium ion in the [Co(η-C5H5)2]1/4TaS2 intercalate, with different dynamic properties in each orientation.

13 C Cross polarization–magic angle spinning spectra have been obtained for crystalline pencillins in which essentially all the resonances have been resolved and assigned. Comparison of the spectra of a series of penicillins of known structure has enabled separation of the effects of intermolecular and intramolecular interactions to be achieved. Two aspects of particular interest have emerged from the work. First, dynamic behaviour of the sidechain is evident in the spectra and this has been shown to depend critically on the crystalline environment of the penicillin molecules. Second, by correlating chemical shifts in the series of molecules, it has proved possible to predict features in the solid-state conformations of penicillins of unknown crystal structure. Further, on comparison with solution spectra, the existence of rapidly interconverting thiazolidine ring puckers in solution is suggested and their relative populations estimated for the different penicillins studied.

Correlations have been observed between the 31P n.m.r. chemical shifts of crystalline inorganic phosphates and the bond strengths at the phosphate oxygen atoms; these provide a basis for structural studies and for peak assignment in phosphates that give more than one resonance.

Whilst the 31P n.m.r. spectra of H2Os3(CO)10L′ and Os3(CO)11L′{L′= PPh2[CH2]2Si(OEt)3} bound to silica are consistent with simple immobilisation, that of materials reported to contain H2Os3(CO)9L′ are inconsistent with this structure.

A method for the separation of pseudo-contact and contact contributions to the shifts of n.m.r. lines has been devised. The method is tested on the 31P resonance of LnIII complexes of cytidine 5′-monophosphate.

Series of experimental data on the n.m.r. line shifts of nuclei in ligands bound to lanthanide cations are presented; the agreement between the observed shifts and those predicted by a new theoretical treatment is excellent, and a procedure for the use of the lanthanides in structural studies, as aqueous cations or in shift reagents is outlined.