Superchiral Plasmonic Phase Sensitivity for Fingerprinting of Protein Interface Structure.

The structure adopted by biomaterials, such as proteins, at interfaces is a crucial parameter in a range of important biological problems. It is a critical property in defining the functionality of cell/bacterial membranes and biofilms (i.e., in antibiotic-resistant infections) and the exploitation of immobilized enzymes in biocatalysis. The intrinsically small quantities of materials at interfaces precludes the application of conventional spectroscopic phenomena routinely used for (bio)structural analysis due to a lack of sensitivity. We show that the interaction of proteins with superchiral fields induces asymmetric changes in retardation phase effects of excited bright and dark modes of a chiral plasmonic nanostructure. Phase retardations are obtained by a simple procedure, which involves fitting the line shape of resonances in the reflectance spectra. These interference effects provide fingerprints that are an incisive probe of the structure of interfacial biomolecules. Using these fingerprints, layers composed of structurally related proteins with differing geometries can be discriminated. Thus, we demonstrate a powerful tool for the bioanalytical toolbox.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Mapping the Aggregation Kinetics of a Therapeutic Antibody Fragment.

The analytical characterization of biopharmaceuticals is a fundamental step in the early stages of development and prediction of their behavior in bioprocesses. Protein aggregation in particular is a common issue as it affects all stages of product development. In the present work, we investigate the stability and the aggregation kinetics of A33Fab, a therapeutically relevant humanized antibody fragment at a wide range of pH, ionic strength, and temperature. We show that the propensity of A33Fab to aggregate under thermally accelerated conditions is pH and ionic-strength dependent with a stronger destabilizing effect of ionic strength at low pH. In the absence of added salts, A33Fab molecules appear to be protected from aggregation due to electrostatic colloidal repulsion at low pH. Analysis by transmission electron microscopy identified significantly different aggregate species formed at low and high pH. The correlations between apparent midpoints of thermal transitions (Tm,app values), or unfolded mole fractions, and aggregation rates are reported here to be significant only at the elevated incubation temperature of 65 °C, where aggregation from the unfolded state predominates. At all other conditions, particularly at 4-45 °C, aggregation of A33 Fab was predominantly from a native-like state, and the kinetics obeyed Arrhenius behavior. Despite this, the rank order of aggregation rates observed at 45 °C, 23 and 4 °C still did not correlate well to each other, indicating that forced degradation at elevated temperatures was not a good screen for predicting behavior at low temperature.

Allergen immobilisation and signal amplification by quantum dots for use in a biosensor assay of IgE in serum.

The production of biosensors for point of care diagnostics usually requires the immobilisation and storage of protein (for example, antigen or antibody) on a sensor surface, in a manner that retains a high degree of activity and low levels of non-specific binding. These characteristics have been assessed for polymer immobilised antigens (allergens) using an IgG binding assay and demonstrated further by assay with serum containing reactive IgEs. The activity of allergens immobilised on sensor chips using copoly(DMA-NAS-MAPS) and a spotting technique, as well as the specificity of their binding interactions with cognate immunoglobulins was assessed using Dual Polarisation Interferometry (DPI). The data obtained indicate that the allergens studied remain stable over long periods of time (at least 114 days). This performance compared favourably with other immobilisation methods. Allergen coated chips were tested in an anti-casein IgE assay using human serum from allergic and non-allergic donors. Detection of both total Ig and specific IgE was demonstrated using a secondary anti-IgE antibody. Furthermore, optical signal enhancement with streptavidin conjugated quantum dots was shown to yield responses for samples below 0.84 ng/mL (0.35 KU/L) of IgE, which overlap with the industrial quasi-standard ImmunoCAP(®) and is the clinically relevant threshold used to classify serum samples from allergic individuals.

Ligand binding to distinct states diverts aggregation of an amyloid-forming protein.

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of ß(2) microglobulin (ß(2)m) fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits ß(2)m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation and diverting the course of assembly to the formation of spherical aggregates. The results demonstrate the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers toward alternative assembly pathways.

Intermolecular alignment in ß2-microglobulin amyloid fibrils.

The deposition of amyloid-like fibrils, composed primarily of the 99-residue protein ß2-microglobulin (ß2m), is one of the characteristic symptoms of dialysis-related amyloidosis. Fibrils formed in vitro at low pH and low salt concentration share many properties with the disease related fibrils and have been extensively studied by a number of biochemical and biophysical methods. These fibrils contain a significant ß-sheet core and have a complex cryoEM electron density profile. Here, we investigate the intrasheet arrangement of the fibrils by means of (15)N-(13)C MAS NMR correlation spectroscopy. We utilize a fibril sample grown from a 50:50 mixture of (15)N,(12)C- and (14)N,(13)C-labeled ß2m monomers, the latter prepared using 2-(13)C glycerol as the carbon source. Together with the use of ZF-TEDOR mixing, this sample allowed us to observe intermolecular (15)N-(13)C backbone-to-backbone contacts with excellent resolution and good sensitivity. The results are consistent with a parallel, in-register arrangement of the protein subunits in the fibrils and suggest that a significant structural reorganization occurs from the native to the fibril state.

Magic angle spinning NMR analysis of beta2-microglobulin amyloid fibrils in two distinct morphologies.

Beta(2)-microglobulin (beta(2)m) is the major structural component of amyloid fibrils deposited in a condition known as dialysis-related amyloidosis. Despite numerous studies that have elucidated important aspects of the fibril formation process in vitro, and a magic angle spinning (MAS) NMR study of the fibrils formed by a small peptide fragment, structural details of beta(2)m fibrils formed by the full-length 99-residue protein are largely unknown. Here, we present a site-specific MAS NMR analysis of fibrils formed by the full-length beta(2)m protein and compare spectra of fibrils prepared under two different conditions. Specifically, long straight (LS) fibrils are formed at pH 2.5, while a very different morphology denoted as worm-like (WL) fibrils is observed in preparations at pH 3.6. High-resolution MAS NMR spectra have allowed us to obtain (13)C and (15)N resonance assignments for 64 residues of beta(2)m in LS fibrils, including part of the highly mobile N-terminus. Approximately 25 residues did not yield observable signals. Chemical shift analysis of the sequentially assigned residues indicates that these fibrils contain an extensive beta-sheet core organized in a non-native manner, with a trans-P32 conformation. In contrast, WL fibrils exhibit more extensive dynamics and appear to have a smaller beta-sheet core than LS fibrils, although both cores seem to share some common elements. Our results suggest that the distinct macroscopic morphological features observed for the two types of fibrils result from variations in structure and dynamics at the molecular level.

Stacked sets of parallel, in-register beta-strands of beta2-microglobulin in amyloid fibrils revealed by site-directed spin labeling and chemical labeling.

beta(2)-microglobulin (beta(2)m) is a 99-residue protein with an immunoglobulin fold that forms beta-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from beta(2)m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other beta(2)m aggregates. A continuous array of parallel, in-register beta-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of beta(2)m consist of about six beta(2)m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like beta-sandwich structure commonly proposed for amyloid fibrils.

Glimpses of the molecular mechanisms of beta2-microglobulin fibril formation in vitro: aggregation on a complex energy landscape.

Beta(2)-microglobulin (beta(2)m) is a 99-residue protein that aggregates to form amyloid fibrils in dialysis-related amyloidosis. The protein provides a powerful model for exploration of the structural molecular mechanisms of fibril formation from a full-length protein in vitro. Fibrils have been assembled from beta(2)m under both low pH conditions, where the precursor is disordered, and at neutral pH where the protein is initially natively folded. Here we discuss the roles of sequence and structure in amyloid formation, the current understanding of the structural mechanisms of the early stages of aggregation of beta(2)m at both low and neutral pH, and the common and distinct features of these assembly pathways.

Competition between intramolecular and intermolecular interactions in an amyloid-forming protein.

Despite much progress in understanding the folding and the aggregation processes of proteins, the rules defining their interplay have yet to be fully defined. This problem is of particular importance since many diseases are initiated by protein unfolding and hence the propensity to aggregate competes with intramolecular collapse and other folding events. Here, we describe the roles of intramolecular and intermolecular interactions in defining the length of the lag time and the apparent rate of elongation of the 100-residue protein human beta(2)-microglobulin at pH 2.5, commencing from an acid-denatured state that lacks persistent structure but contains significant non-random hydrophobic interactions. Using a combination of site-directed mutagenesis, quantitative kinetic analysis and computational methods, we show that only a single region of about 10 residues in length, determines the rate of fibril formation, despite the fact that other regions exhibit a significant intrinsic propensity for aggregation. We rationalise these results by analysing the effect of incorporating the conformational properties of acid-unfolded beta(2)-microglobulin and its variants at pH 2.5 as measured by NMR spectroscopy into the Zyggregator aggregation prediction algorithm. These results demonstrate that residual structure in the precursor state modulates the intrinsic propensity of the polypeptide chain to aggregate and that the algorithm developed here allows the key regions for aggregation to be more clearly identified and the rates of their self-association to be predicted. Given the common propensity of unfolded chains to form non-random intramolecular interactions as monomers and to self-assemble subsequently into amyloid fibrils, the approach developed should find widespread utility for the prediction of regions important in amyloid formation and their rates of self-assembly.

Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation.

Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human beta(2)-microglobulin (beta(2)m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type beta(2)m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the beta(2)m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29-51 and 58-79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for beta(2)m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62-70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.

Towards an understanding of the structural molecular mechanism of beta(2)-microglobulin amyloid formation in vitro.

Deriving a complete understanding of protein self-association into amyloid fibrils across multiple distance and time scales is an enormous challenge. At small length scales, a detailed description of the partially folded protein ensemble that participates in self-assembly remains obscure. At larger length scales, amyloid fibrils are often heterogeneous, can form along multiple pathways, and are further complicated by phenomena such as phase-separation. Over the last 5 years, we have used an array of biophysical approaches in order to elucidate the structural and molecular mechanism of amyloid fibril formation, focusing on the all beta-sheet protein, beta(2)-microglubulin (beta(2)m). This protein forms amyloid deposits in the human disease 'dialysis-related amyloidosis' (DRA). We have shown that under acidic conditions beta(2)m rapidly associates in vitro to form amyloid-like fibrils that have different morphological properties, but which contain an underpinning cross-beta structure. In this review, we discuss our current knowledge of the structure of these fibrils, as well as the structural, kinetic and thermodynamic relationship between fibrils with different morphologies. The results provide some of the first insights into the shape of the self-assembly free-energy landscape for this protein and highlight the parallel nature of the assembly process. We include a detailed description of the structure and dynamics of partially folded and acid unfolded species of beta(2)m using NMR, and highlight regions thought to be important in early self-association events. Finally, we discuss briefly how knowledge of assembly mechanisms in vitro can be used to inform the design of therapeutic strategies for this, and other amyloid disorders, and we speculate on how the increasing power of biophysical approaches may lead to a fuller description of protein self-assembly into amyloid in the future.

Extending the folding nucleus of ubiquitin with an independently folding beta-hairpin finger: hurdles to rapid folding arising from the stabilisation of local interactions.

The N-terminal beta-hairpin sequence of ubiquitin has been implicated as a folding nucleation site. To extend and stabilise the ubiquitin folding nucleus, we have inserted an autonomously folding 14-residue peptide sequence beta4 which in isolation forms a highly populated beta-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Ubeta4) shows that the hairpin finger is fully structured and stabilises ubiquitin by approximately 8kJmol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of Ubeta4 mutants shows that the hairpin extension of Ubeta4 is also significantly populated in the transition state (phi(F)-values >0.7) and has the effect of templating the formation of native contacts in the folding nucleus of ubiquitin. However, at low denaturant concentrations the chevron plot of Ubeta4 shows a small deviation from linearity (roll-over effect), indicative of the population of a compact collapsed state, which appears to arise from over-stabilisation of local interactions. Destabilising mutations within the native hairpin sequence and within the engineered hairpin extension, but not elsewhere, eliminate this non-linearity and restore apparent two-state behaviour. The pitfall to stabilising local interactions is to present hurdles to the rapid and efficient folding of small proteins down a smooth folding funnel by trapping partially folded or misfolded states that must unfold or rearrange before refolding.

Dynamics in the unfolded state of beta2-microglobulin studied by NMR.

Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.

Context-dependent effects of proline residues on the stability and folding pathway of ubiquitin.

Substitution of trans-proline at three positions in ubiquitin (residues 19, 37 and 38) produces significant context-dependent effects on protein stability (both stabilizing and destabilizing) that reflect changes to a combination of parameters including backbone flexibility, hydrophobic interactions, solvent accessibility to polar groups and intrinsic backbone conformational preferences. Kinetic analysis of the wild-type yeast protein reveals a predominant fast-folding phase which conforms to an apparent two-state folding model. Temperature-dependent studies of the refolding rate reveal thermodynamic details of the nature of the transition state for folding consistent with hydrophobic collapse providing the overall driving force. Brønsted analysis of the refolding and unfolding rates of a family of mutants with a variety of side chain substitutions for P37 and P38 reveals that the two prolines, which are located in a surface loop adjacent to the C terminus of the main alpha-helix (residues 24-33), are not significantly structured in the transition state for folding and appear to be consolidated into the native structure only late in the folding process. We draw a similar conclusion regarding position 19 in the loop connecting the N-terminal beta-hairpin to the main alpha-helix. The proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways.

Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state.

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.