Susie J. Meade

The role of dicarbonyl compounds in non-enzymatic crosslinking: a structure-activity study.

The Maillard reaction is a complex network of reactions that has been shown to result in the non-enzymatic crosslinking of proteins. Recent attention has focussed on the role of alpha-dicarbonyl compounds as important in vivo contributors to protein crosslinking but, despite extensive research, the molecular mechanisms of the crosslinking reaction remain open to conjecture. In particular, no relationship between the structure of the carbonyl-containing compounds and their activity as crosslinking agents has been established. In an effort to elucidate a structure-reactivity relationship, a wide range of dicarbonyl compounds, including linear, cyclic, di-aldehyde and di-ketone compounds, were reacted with the model protein ribonuclease A and their crosslinking activity assessed. Methylglyoxal and glutaraldehyde were found to be the most efficient crosslinkers, whilst closely related molecules effected crosslinking at a much lower rate. Cyclopentan-1,2-dione was also shown to be a reactive crosslinking agent. The efficiency of methylglyoxal and glutaraldehyde at crosslinking is thought to be related to their ability to form stable heterocyclic compounds that are the basis of protein crosslinks. The reasons for the striking reactivity of these two compounds, compared to closely related structures is explained by subtle balances between competing pathways in a complex reaction network.

Amyloid fibrils as a nanoscaffold for enzyme immobilization

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross‐linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD‐functionalized amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold.

Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: Towards bioremediation and chemical detoxification

Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (∼300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization.

New insights into protein crosslinking via the Maillard reaction: structural requirements, the effect on enzyme function, and predicted efficacy of crosslinking inhibitors as anti-ageing therapeutics.

Protein crosslinking via the Maillard reaction with alpha-dicarbonyl compounds has been the subject of intense literature scrutiny. We report here a systematic study of three previously-neglected aspects of the reaction. Firstly, structural requirements were probed. An arginine-free peptide that contains two lysine residues, and a lysine-free peptide that contains arginine, were reacted with glyoxal, methylglyoxal and biacetyl. Methylglyoxal was able to crosslink in the absence of arginine residues, but glyoxal and biacetyl were not. Glyoxal crosslinked the lysine-free peptide via the N-terminus, but methylglyoxal and biacetyl could not. In this study, crosslinking did not require the presence of arginine but did require a free amino group, from a lysine residue, or the N-terminus. Thus specificity in structural requirements for protein crosslinking by alpha-dicarbonyls has been demonstrated. Secondly, protein function following glycation was examined by treating ribonuclease A with the three alpha-dicarbonyls, which were shown both to crosslink the enzyme and impair enzymatic activity. Thirdly, the effects of two reported Maillard reaction inhibitors, aminoguanidine and 3,5-dimethylpyrazole-1-carboxamidine on the crosslinking reaction were assessed, with a parallel measurement of the effect on enzyme activity. The results demonstrate that preventing protein crosslinking does not necessarily preserve enzyme activity. These results cast doubt on the likely efficacy of some purported anti-ageing compounds in vivo.

Crosslinkage of proteins by dehydroascorbic acid and its degradation products

Abstract Protein crosslinking can have a profound effect on the structure and function of proteins in food. Dehydroascorbic acid (DHA) has been shown to be involved in Maillard type chemistry that leads to protein crosslinking. In this study, the effect of temperature on the rate of this reaction was studied. The reaction was shown to proceed rapidly at temperatures that may be encountered during food processing. In order to assess the relative reactivity of DHA and its breakdown products, five known degradation products were reacted with protein and their crosslinking ability, via Maillard chemistry, was assessed. Oxalic acid did not effect protein crosslinking. Threose, glyoxal, diacetyl and methyl glyoxal all reacted faster than DHA. The main crosslinking reaction observed was shown to involve a lysine residue. Our results suggest that these molecules may be important in determining the modification of protein functionality during food processing.

Protein nanofibres of defined morphology prepared from mixtures of crude crystallins

Self assembling protein nanofibres have great potential within the nanotechnology industry. To realise this potential, nanofibres need to be manufactured on a commercial scale. To date, amyloid fibrils, a form of protein nanofibre with desirable bionanotechnological properties, have only been produced using expensive and time-costly, small-scale methods. This paper describes the production of protein nanofibres from semi-pure and crude mixtures of bovine crystallin proteins, currently a waste product in the meat industry. These nanofibres displayed amyloid fibril characteristics. Moreover, distinct fibre morphologies were observed dependent upon the formation conditions adopted. This research shows that amyloid fibrils can be inexpensively produced from crude crystallin protein mixtures, making them a potential industrial source for protein nanofibres.

Protein Cross‐Linking in Food

Abstract: The aims of this paper are (1) to probe the relationship between molecular structure and protein cross‐linking ability for a range of small molecules; (2) to establish whether this relationship holds within a food matrix; and (3) to test the impact of Maillard cross‐linking on food functionality, particularly texture, in wheat‐ and soy‐based food systems. A variety of molecules were obtained, either commercially or via organic synthesis. Cross‐linking ability was tested using our standard model system, employing ribonuclease A and analyzing the results by SDS‐PAGE. Molecules of varying reactivity were tested in wheat‐ and soy‐based products, and the changes in functionality were correlated with changes in protein cross‐linking. No simple relationship was found between molecular structure and ability to cross‐link ribonuclease. Only the most reactive reagents were able to cross‐link within the food matrix. Nevertheless, a low degree of cross‐linking was shown to have significant consequences on the properties of wheat‐ and soy‐based foods, suggesting that the Maillard reaction may represent a means to control food texture.

Amyloid fibrils as functionalizable components of nanocomposite materials

Amyloid fibrils are a form of protein nanofiber that show promise as components of multifunctional bionanomaterials. In this work, native bovine insulin and bovine insulin that had been previously converted into amyloid fibrils were combined with poly(vinyl alcohol) (PVOH) via solution casting to determine the effect of fibrillization on the thermomechanical properties of the resulting composite. The synthesis method was found to preserve the amyloid fibril structure and properties of the resulting fibril‐PVOH composite were investigated. At a filling level of 0.6 wt %, the fibril‐reinforced PVOH was 15% stiffer than the PVOH control. Various properties of the films, including the glass transition temperature, degradation temperature, microstructure, and film morphology were characterized. Although more work is required to optimize the properties of the composites, this study provides proof of principle that incorporation of amyloid fibrils into a polymeric material can impart useful changes to the mechanical and morphological properties of the films.

Controlling the dimensions of amyloid fibrils: Toward homogenous components for bionanotechnology

Amyloid fibrils have been recognized as having potential in a variety of bionanotechnological applications. However, realization of these applications is constrained by a lack of control over morphology and alignment, both crucial for potential end uses. This article focuses on the use of growth and storage conditions to control the length of amyloid fibrils formed from bovine insulin, with length distributions constructed from transmission electron microscopy (TEM) images. Growth temperature, pH, protein concentration, and storage conditions were examined and were seen to offer a range of conditions that favor different length distribution. The use of amyloid fibrils as nanowires is one area where control of fibril dimensions is desirable, for experimental setup and endpoint applications. The conductive properties of fibrils formed from bovine insulin are presented, with these insulin fibrils being shown to have high resistivity in their unmodified state, with current values in the nanoamp range. These low current values can be increased via modification, or the fibrils used in their native state in applications where low current values are desirable. These findings, coupled with the ability to predict and select for various insulin amyloid fibril dimensions, enhances their utility as nanomaterials.

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid‐forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of “wavy” fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom‐up assembly of higher order protein structures from amyloid fibrils.