The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and [alpha]-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a [beta]-sheet arrangement reminiscent of the [beta]-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme.

The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97-Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis

Small-Angle X-ray Scattering Screening Complements Conventional Biophysical Analysis: Comparative Structural and Biophysical Analysis of Monoclonal Antibodies IgG1, IgG2, and IgG4

ABSTRACTA crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development. © 2014 The Authors. Journal of Pharmaceutical Sciences published by Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1701-1710, 201

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97–Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.</jats:p

Recommendations for addressing the translational gap between experimental and clinical research on amyloid diseases

This paper is a report of recommendations for addressing translational challenges in amyloid disease research. They were developed during and following an international online workshop organized by the LINXS Institute of Advanced Neutron and X-Ray Science in March 2021. Key suggestions include improving cross-cultural communication between basic science and clinical research, increasing the influence of scientific societies and journals (vis-à-vis funding agencies and pharmaceutical companies), improving the dissemination of negative results, and strengthening the ethos of science

The structural basis of fungal glucuronoyl esterase activity on natural substrates

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/β-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry

The structural basis of fungal glucuronoyl esterase activity on natural substrates

AbstractStructural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/β-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.</jats:p

Distinct α-Synuclein:Lipid Co-Structure Complexes Affect Amyloid Nucleation through Fibril Mimetic Behavior

A hallmark of Parkinson’s disease is the presence of Lewy bodies consisting of lipids and proteins, mainly fibrillated α-synuclein (aSN). aSN is an intrinsically disordered protein exerting its physiological role in an ensemble of states, one of which coexists in large assemblies with lipids, recently termed co-structures. Here, we decipher the kinetics of aSN:lipid co-structure formation to decode its mechanism of formation, and we show that the co-structures form with a distinct stoichiometry. Through seeded fibrillation assays, we demonstrate that aSN:lipid co-structures accelerate aSN fibril nucleation compared to lipid vesicles alone. A small-angle X-ray scattering-based model is proposed in which aSN decorates the lipid vesicle surface, yielding properties similar to those of the fibril surface, enhancing fibril nucleation. The delicate balance of aSN structural states close to and on the membrane may under given conditions, e.g., increased local concentrations, be a crucial switching factor between functional and pathological behavior