The Fluorescent Amino Acid p‐Cyanophenylalanine Provides an Intrinsic Probe of Amyloid Formation

Amyloid formation has been implicated in more than fifteen different human diseases including Alzheimer’s disease, Parkinson’s disease, prion-based diseases, and type 2 diabetes. 2] The kinetics of amyloid formation are complex, and typically consist of a lag phase during which little fibrillar material is produced followed by a rapid growth phase. Characterization of the kinetics of amyloid formation and the nature of any intermediates that are formed have emerged as critical topics in the field since there is growing evidence that prefibrillar intermediate structures might be the toxic species. Unfortunately, a limited set of low resolution spectroscopic methods can be applied to study the kinetics of amyloid formation and residue-specific information is generally not obtainable. Amyloid formation, in vitro, is traditionally followed by fluorescence detection in thioflavin T-binding experiments. The fluorescence of the dye significantly increases upon binding to the amyloid fibril. The assay is simple to execute, however, it does suffer from some noticeable drawbacks. First, the exact mechanism for the fluorescence enhancement is not completely understood, hence, it is not completely clear what the dye binding probes. Second, the dye does not bind to prefibrillar intermediates and thus cannot be used to follow their formation. A third extremely important but somewhat subtle issue involves the study of inhibitors. Some compounds can bind to amyloid fibrils and displace bound thioflavin T without inhibiting amyloid formation. In these cases thioflavin T assays lead to the incorrect conclusion that the compound is an amyloid inhibitor. Fourth, the dye is an extrinsic probe and there is always the risk that the kinetics of assembly could be affected since the assay is conducted by adding the dye to the peptide solution and it binds to the fibrils as they are being formed. In principle, intrinsic protein fluorescence could be used to follow amyloid formation since Trp fluorescence is sensitive to the local environment. However, a surprising number of important amyloidogenic polypeptides lack Trp including Ab, a-synuclein, and IAPP (amylin), the causative agents of amyloid formation in Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes, respectively. Furthermore, the addition of Trp by mutagenesis often represents a nonconserved mutation. Tyr fluorescence might be useful, but it is less sensitive than Trp fluorescence and its interpretation is much less straightforward. It would clearly be desirable to have access to another fluorescent amino acid that could be used as a probe of amyloid formation. An ideal amino-acid analogue should exhibit a large, easily interpretable change in fluorescence during the process of amyloid formation, but represent only a small perturbation on the structure and hydrophobicity of one or more of the twenty genetically encoded residues, and thus allow for conservative substitution. p-Cyanophenyalanine (p-cyanoPhe) appears to meet all of these requirements. Its fluorescence quantum yield is very sensitive to solvent interactions and is decreased significantly in a hydrophobic environment compared to its value in water ; this makes it a sensitive probe of the local environment. Importantly, it has a blue-shifted absorption band, which allows its fluorescence to be selectively excited in the presence of Tyr or Trp. The cyano group is a hydrogen-bond acceptor, but it has the very desirable feature that it is readily accommodated in the hydrophobic core of proteins since its polarity is intermediate between that of an amide and a methylene. It is also considerably smaller than Trp, which makes it a very conservative replacement for either Phe or Tyr. In the present work we demonstrate the use of p-cyanoPhe fluorescence to probe amyloid formation using islet amyloid polypeptide (IAPP, amylin) as a test case. IAPP is responsible for the formation of pancreatic islet amyloid in type 2 diabetes. Islet amyloid formation plays a role in the pathology of the disease by killing pancreatic b cells, and contributing to the loss of b cell mass and the decline in insulin secretion. IAPP is 37 residues in length, contains a disulfide bond that links residues 2 and 7, and has an amidated C terminus. It does not contain Trp, but does have two Phe residues at positions 15 and 23 and a single Tyr at its C terminus. We replaced Tyr37 with p-cyanoPhe. The peptide is denoted hIAPP-Y37FC N. The sequence of the wild-type human peptide—denoted here hIAPP—is: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY