Residue-Specific, Real-Time Characterization of Lag-Phase Species and Fibril Growth During Amyloid Formation: A Combined Fluorescence and IR Study of p-Cyanophenylalanine Analogs of Islet Amyloid Polypeptide

Abstract

Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (FC≡N) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by FC≡N. The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. FC≡N fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and FC≡N fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until β-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. FC≡N also provides information about fibril structure. Fluorescence emission measurements, infrared measurements, and quenching studies indicate that the aromatic residues are differentially exposed in the fibril state with Phe15 being the most exposed. FC≡N is readily accommodated into proteins; thus, the approach should be broadly applicable.

Matrix Metalloproteinase-9 Protects Islets from Amyloid-induced Toxicity

“…The fluorescence of p-cyanoPhe is sensitive to environmental changes and can be used to monitor the kinetics of fibril formation. This substitution does not perturb the kinetics of amyloid formation and the measured time course of fibril formation is identical to the one probed using wild-type hIAPP (23,24). hIAPP-F15 p-cyanoPhe can therefore be used to monitor fibril formation by full-length hIAPP in the presence of another amyloid forming peptide (e.g.…”

Sensing pH via p-cyanophenylalanine fluorescence: Application to determine peptide pKa and membrane penetration kinetics

“…The broad spectroscopic utility of this unnatural amino acid, which is a structural derivative of tyrosine (Tyr) or phenylalanine (Phe) [2], stems from the fact that its fluorescence quantum yield and lifetime are sensitive to environment [3], as well as its easy incorporation into peptides and proteins [4]. For example, dehydration leads to a significant decrease in the fluorescence intensity of Phe CN , thus making it a useful probe of processes that involve exclusion of water, such as protein folding [5,6], binding [7,8], aggregation [9–11], and interaction with membranes [12–15]. In addition, the fluorescence quantum yield of Phe CN can be modulated by various metal ions [16] and several amino acid sidechains [17–21].…”

Manipulation of enzyme properties by noncanonical amino acid incorporation

“…Second, a NCAA incorporated into a protein can serve as a probe of protein folding, protein-protein interactions, and ligand binding to proteins. The nitrile group of p-cyanophenylalanine has been used as a fluorescence and infrared probe to study ligand binding to myoglobin, folding of ribosomal protein L9, and aggregation of islet amyloid polypeptide [13][14][15]. Incorporating NCAA can also facilitate NMR spectroscopic studies of proteins (reviewed in [16]).…”