Juliet A. Gerrard

Volta phase plate cryo-EM of the small protein complex Prx3

Cryo-EM of large, macromolecular assemblies has seen a significant increase in the numbers of high-resolution structures since the arrival of direct electron detectors. However, sub-nanometre resolution cryo-EM structures are rare compared with crystal structure depositions, particularly for relatively small particles (<400u2009kDa). Here we demonstrate the benefits of Volta phase plates for single-particle analysis by time-efficient cryo-EM structure determination of 257u2009kDa human peroxiredoxin-3 dodecamers at 4.4u2009Å resolution. The Volta phase plate improves the applicability of cryo-EM for small molecules and accelerates structure determination.

Cryo-electron microscopy structure of human peroxiredoxin-3 filament reveals the assembly of a putative chaperone.

Peroxiredoxins (Prxs) are a ubiquitous class of thiol-dependent peroxidases that play an important role in the protection and response of cells to oxidative stress. The catalytic unit of typical 2-Cys Prxs are homodimers, which can self-associate to form complex assemblies that are hypothesized to have signaling and chaperone activity. Mitochondrial Prx3 forms dodecameric toroids, which can further stack to form filaments, the so-called high-molecular-weight (HMW) form that has putative holdase activity. We used single-particle analysis and helical processing of electron cryomicroscopy images of human Prx3 filaments induced by low pH to generate a ∼7-Å resolution 3D structure of the HMW form, the first such structure for a 2-Cys Prx. The pseudo-atomic model reveals interactions that promote the stacking of the toroids and shows that unlike previously reported data, the structure can accommodate a partially folded C terminus. The HMW filament lumen displays hydrophobic patches, which we hypothesize bestow holdase activity.

Structures of Human Peroxiredoxin 3 Suggest Self-Chaperoning Assembly that Maintains Catalytic State

Peroxiredoxins are antioxidant proteins primarily responsible for detoxification of hydroperoxides inxa0cells. On exposure to various cellular stresses, peroxiredoxins can acquire chaperone activity, manifested as quaternary reorganization into a high molecular weight (HMW) form. Acidification, for example, causes dodecameric rings of human peroxiredoxin 3 (HsPrx3) to stack into long helical filaments. In this work, a 4.1-Å resolution structure of low-pH-instigated helical filaments was elucidated, showing a locally unfolded active site and partially folded C terminus. A 2.8-Å crystal structure of HsPrx3 was determined at pH 8.5 under reducing conditions, wherein dodecameric rings are arranged as a short stack, with symmetry similar to low-pH filaments. In contrast to previous observations, the crystal structure displays both a fully folded active site and ordered C terminus, suggesting that the HsPrx3 HMW form maintains catalytic activity. We propose a new role for the HMW form as a self-chaperoning assembly maintaining HsPrx3 function under stress.

Controlling gelation with sequence: towards programmable peptide hydrogels

UNLABELLEDnThe self-assembling peptide IKHLSVN, inspired by inspection of a protein-protein interface, has previously been reported as one of a new class of bio-inspired peptides. Here the peptide, dubbed littleSven, and modifications designed to probe the resilience of the sequence to self-assembly, is characterised. Although the parent peptide did not form a hydrogel, small modifications to the sequence (one side chain or an N-terminus modification) led to hydrogels with properties (eg. gelation time and rheology) that could be tuned by these small alterations. The results suggest that peptides derived from protein-protein interfaces are resilient to changes in sequence and can be harnessed to form hydrogels with controlled properties.nnnSTATEMENT OF SIGNIFICANCEnNatural occurring self-assembly peptides are attractive building blocks for engineered bionanomaterials due to their biocompatibility and biodegradability. The bio-inspired self-assembly peptide, IKHLSVN, was used as a template to design peptides that readily formed hydrogels. The peptide sequence was specifically tuned to create a bionanomaterial with different properties that could be exploited downstream for a broad range of applications: nanowires, drug release, vaccine adjuvant, tissue engineering. We describe how small modifications to the parent peptide alter the amyloid-like characteristics and gel strength for each peptide.

Quaternary structure influences the peroxidase activity of peroxiredoxin 3

Peroxiredoxins are abundant peroxidase enzymes that are key regulators of the cellular redox environment. A major subgroup of these proteins, the typical 2-Cys peroxiredoxins, can switch between dimers and decameric or dodecameric rings, during the catalytic cycle. The necessity of this change in quaternary structure for function as a peroxidase is not fully understood. In order to explore this, human peroxiredoxin 3 (Prx3) protein was engineered to form both obligate dimers (S75E Prx3) and stabilised dodecameric rings (S78C Prx3), uncoupling structural transformations from the catalytic cycle. The obligate dimer, S75E Prx3, retained catalytic activity towards hydrogen peroxide, albeit significantly lower than the wildtype and S78C proteins, suggesting an evolutionary advantage of having higher order self-assemblies.

Novel lift-off technique for Transmission Electron Microscopy imaging of block copolymer films.

We have developed a simple technique to allow for the lift-off and subsequent transfer of poly(styrene-block-ethylene glycol) films to Transmission Electron Microscopy (TEM) grids. The block copolymer is spin coated onto carbon coated mica and annealed. After the thin film is produced it can easily be floated onto water and picked up by a TEM grid. This method offers better control over film processing than dip coating the TEM grid and is also a significant improvement over methods using etchants such as hydrofluoric acid.

Functional Amyloid Protection in the Eye Lens: Retention of α-Crystallin Molecular Chaperone Activity after Modification into Amyloid Fibrils

Amyloid fibril formation occurs from a wide range of peptides and proteins and is typically associated with a loss of protein function and/or a gain of toxic function, as the native structure of the protein undergoes major alteration to form a cross β-sheet array. It is now well recognised that some amyloid fibrils have a biological function, which has led to increased interest in the potential that these so-called functional amyloids may either retain the function of the native protein, or gain function upon adopting a fibrillar structure. Herein, we investigate the molecular chaperone ability of α-crystallin, the predominant eye lens protein which is composed of two related subunits αA- and αB-crystallin, and its capacity to retain and even enhance its chaperone activity after forming aggregate structures under conditions of thermal and chemical stress. We demonstrate that both eye lens α-crystallin and αB-crystallin (which is also found extensively outside the lens) retain, to a significant degree, their molecular chaperone activity under conditions of structural change, including after formation into amyloid fibrils and amorphous aggregates. The results can be related directly to the effects of aging on the structure and chaperone function of α-crystallin in the eye lens, particularly its ability to prevent crystallin protein aggregation and hence lens opacification associated with cataract formation.

MALDI-imaging enables direct observation of kinetic and thermodynamic products of mixed peptide fiber assembly

Controlling the self-assembly of multicomponent systems provides a key to designing new materials and understanding the molecular complexity of biology. Here, we demonstrate the first use of MALDI-imaging to characterize a multicomponent self-assembling peptide fiber. Observations of mixed peptide systems over time demonstrate how simple sequence variation can change the balance between kinetic and thermodynamic products.

Amyloid Fibrils from Hemoglobin

Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of β-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-β quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (−20 to 80 °C) and pHs (2–10), and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products.

Formation of supramolecular protein structures on gold surfaces

Recent research has highlighted the exciting possibilities enabled by the use of protein structures as nanocomponents to form functional nanodevices. To this end, control over protein-protein and protein-surface interactions is essential. In this study, the authors probe the interaction of human peroxiredoxin 3 with gold surfaces, a protein that has been previously identified as having potential use in nanotechnology. Analytical ultracentrifugation and transmission electron microscopy revealed the pH mediated assembly of protein toroids into tubular structures across a small pH range. Quartz crystal microbalance with dissipation measurements showed differences in absorbed protein mass when pH is switched from pH 8.0 to 7.2, in line with the formation of supramolecular structures observed in solution studies. Scanning tunneling microscopy under ambient conditions showed that these protein tubes form on surfaces in a concentration dependent manner, with a tendency for protein adsorption and supramolecular assembly at the edges of Au(111) terraces. Finally, self-assembled monolayer modification of Au surfaces was explored as a means to control the adsorption and orientation of pH triggered protein structures.