Abstract
Relatively short amino acid sequences often play a pivotal role in triggering protein aggregation leading to the formation of amyloid fibrils. In the case of insulin, various regions of A- and B-chains have been implicated as the most relevant to the protein’s amyloidogenicity. Here, we focus on the highly amyloidogenic H-fragment of insulin comprising the disulfide-bonded N-terminal parts of both chains. Analysis of the aggregation behavior of single-chain peptide derivatives of the H-fragment suggests that the A-chain’s part initiates the aggregation process while the disulfide-tethered B-chain reluctantly adapts to amyloid structure. Merging of both A- and B-parts into single-chain continuous peptides (A–B and B–A) results in extreme amyloidogenicity exceeding that of the double-chain H-fragment as reflected by almost instantaneous de novo fibrillization. Amyloid fibrils of A–B and B–A present distinct morphological and infrared traits and do not cross-seed insulin. Our study suggests that the N-terminal part of insulin’s A-chain containing the intact Cys6–Cys11 intrachain disulfide bond may constitute insulin’s major amyloid stretch which, through its bent conformation, enforces a parallel in-register alignment of β-strands. Comparison of the self-association behavior of H, A–B, and B–A peptides suggests that A-chain’s N-terminal amyloid stretch is very versatile and adaptive to various structural contexts.
Highlights
Nowadays, formation of amyloid fibrils is recognized as a generic process accessible to various proteins and peptides.[1,2]Due to association with a number of degenerative maladies (e.g., Alzheimer’s disease, type 2 diabetes mellitus3−5), as well as benign biological functions essential for numerous organisms,[6−8] research on amyloidogenesis has expanded considerably in recent years
Formation of thermodynamically stable fibrillar structures imposes specific requirements on amino acid sequence, backbone topology, and conformation of aggregating proteins.[9−11] For example, β-sheet-breaking proline residues, uncompensated Coulombic repulsion between ionized side chains, and a main-chain topology restricted by numerous disulfide bonds are all intuitively expected to decrease the tendency to form fibrils
It has been shown that protein amyloidogenicity may, adapt well to the presence of proline residues at some sites in the sequences[13] while marginally uncompensated electric charges may even enhance aggregation;[14] there is evidence that disulfide bonds may accelerate formation of fibrils in specific cases.[15−17] the actual impact of even such elementary factors on a protein’s propensity to form fibrils depends on the structural and thermodynamic context
Summary
Formation of amyloid fibrils is recognized as a generic process accessible to various proteins and peptides.[1,2]. It has been elegantly demonstrated by Minor and Kim that whether a particular main-chain segment within a globular protein adopts β-sheet conformation depends strongly on the tertiary context, as opposed to being determined solely by the intrinsic secondary structure preference.[18,19] From this it follows that an actual amyloidogenic propensity of a given amino acid sequence depends on the quaternary context; i.e., its accurate estimation would require a priori knowledge of the 3D structure of the fibril Despite these fundamental challenges, computational algorithms aimed at predicting amyloidogenic tendencies of amino acid sequences (and even of whole folded proteins, e.g., refs 11, 20−24; for a concise review, see ref 10) have been developed with some success.
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