Residue Polypeptide Research Articles

Treatment of diet-induced lipodystrophic C57BL/6J mice with long-acting PASylated leptin normalises insulin sensitivity and hepatic steatosis by promoting lipid utilisation.

Recombinant leptin offers a viable treatment for lipodystrophy (LD) syndromes. However, due to its short plasma half-life, leptin replacement therapy requires at least daily subcutaneous (s.c.) injections. Here, we optimised this treatment strategy in LD mice by using a novel leptin version with extended plasma half-life using PASylation technology. A long-acting leptin version was prepared by genetic fusion with a 600 residue polypeptide made of Pro, Ala and Ser (PASylation), which enlarges the hydrodynamic volume and, thus, retards renal filtration, allowing less frequent injection. LD was induced in C57BL/6J mice by feeding a diet supplemented with conjugated linoleic acid (CLA). Chronic and acute effects of leptin treatment were assessed by evaluating plasma insulin levels, insulin tolerance, histological liver sections, energy expenditure, energy intake and body composition. In a cohort of female mice, 4nmol PAS-leptin (applied via four s.c. injections every 3days) successfully alleviated the CLA-induced LD phenotype, which was characterised by hyperinsulinaemia, insulin intolerance and hepatosteatosis. The same injection regimen had no measurable effect when unmodified recombinant leptin was administered at an equivalent dose. In a cohort of LD males, a single s.c. injection of PAS-leptin did not affect energy expenditure but inhibited food intake and promoted a shift in fuel selection towards preferential fat oxidation, which mechanistically substantiates the metabolic improvements. The excellent pharmacological properties render PASylated leptin an agent of choice for refining both animal studies and therapeutic strategies in the context of LD syndromes and beyond.

Molecular characteristics of three thymosin-repeat proteins from Marsupenaeus japonicus and their responses to WSSV infection

β-thymosins, a family of highly conserved peptides, play a vital role in wound-healing, angiogenesis, antimicrobial process and antiviral immunity. Three novel β-thymosin-repeat proteins, named mjthm4, mjthm3 and mjthm2, were cloned from Marsupenaeus japonicus using expressed sequence tags (EST) from suppression subtractive hybridization. The mjthm4, mjthm3 and mjthm2 cDNAs possessed open reading frames that encoded 166, 128 and 90 amino acid residue polypeptides and contained four, three and two β-thymosin actin binding modules, respectively. Blast analysis demonstrated that mjthm4, mjthm3 and mjthm2 shared high homology with known invertebrate multi-repeat β-thymosins. These proteins are ubiquitously expressed in all of the examined tissues, and the transcriptional levels were highest in the intestine. Further investigation revealed that mjthm4, mjthm3 and mjthm2 were remarkably up-regulated 6 h after WSSV infection. Moreover, while mjthm4 transcriptional levels displayed no changes, mjthm3 and mjthm2 levels decreased in the virus-resistant shrimps. The results indicate that mjthm4, mjthm3 and mjthm2 are novel multi-repeat β-thymosin homologues, have a close relationship with WSSV infection, and might contribute to a better understanding of host defense and/or virus invasion interactions in shrimps.

Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum

Histoplasma capsulatum is the causative agent of human histoplasmosis, which can cause respiratory and systemic mycosis in immune-compromised individuals. Lysine acetylation, a protein posttranslational protein modification, is widespread in both eukaryotes and prokaryotes. Although increasing evidence suggests that lysine acetylation may play critical roles in fungus physiology, very little is known about its extent and function in H. capsulatum. To comprehensively profile protein lysine acetylation in H. capsulatum, we performed a global acetylome analysis through peptide prefractionation, antibody enrichment, and LC−MS/MS analysis, identifying 775 acetylation sites on 456 acetylated proteins; and functionally analysis showing their involvement in different biological processes. We defined six types of acetylation site motifs, and the results imply that lysine residue of polypeptide with tyrosine at the −1 and +1 positions, histidine at the +1 position, and phenylalanine (F) at the +1 and +2 position is a preferred substrate of lysine acetyltransferase. Moreover, some virulence factors candidates including calmodulin and DnaK are acetylated. In conclusion, our data set may serve as an important resource for the elucidation of associations between functional protein lysine acetylation and virulence in H. capsulatum.

Biomimetic design: a programmed tetradecapeptide folds and auto-dimerizes as a stereochemically articulated receptor protein

Treating protein-structure evolution as a hierarchy of selections, a fourteen residue polypeptide was made as a C2 symmetric receptor structure in mimicry of HIV protease. This shows the value of a biomimetic algorithm and of stereochemistry as a variable in protein design.

Fluorescence dynamics of N-terminal Trp–Trp residues in polypeptide: intrinsic indicators for monitoring pH

pH plays a vital role in various cellular activities, and real-time observation of the intracellular pH through a pH indicator is very important for studying many physiological processes. In this paper, we studied the pH response of Trp–Trp dipeptide and its derivatives (NATrp2Me, NBTrp2 and Trp2Me) by steady-state and time-resolved fluorescence spectroscopy. Both the fluorescence intensities and lifetimes of Trp–Trp dipeptide as well as Trp2Me were functions of pH in the physiological range from 5.5 to 9.0. However, NATrp2Me and NBTrp2 showed no difference. The exposed amino was found to be pivotal for its pH dependence. Moreover, an artificially synthesized tetrapeptide (Trp–Trp–Ala–Ser) confirmed the pH sensitivity of N-terminal Trp–Trp residues. The pH values could be quantitatively determined from the fluorescence intensities and lifetimes of the N-terminal Trp–Trp residue. Thus, the N-terminal Trp–Trp residues may be fused into the polypeptides/proteins to serve as an intrinsic pH indicator in fluorescence spectroscopy and imaging.

Alkyl hydroperoxide reductase from Bacillus aquimaris MKSC 6.2 protects Esherichia coli from oxidative stress.

Alkyl hydroperoxide reductase genes (ahpCF) from the soft coral associated Bacillus aquimaris MKSC6.2 have been isolated. The cloned 546 bp ahpC gene encodes a 181 amino acid residues polypeptide. The AhpC belongs to typical 2-Cys peroxiredoxin (Prx) containing conserved peroxidatic cysteine residue (C46 ) required for hydroperoxide reduction and conserved resolving cysteine (C166 ). The isolated 1530 bp ahpF gene encodes a polypeptide of 509 amino acid residues with two conserved C128 HNC131 and C337 PHC340 catalytic residues required for reduction of oxidized-AhpC during catalytic turnover. A survival study with Escherichia coli showed that overexpression of AhpC and AhpF resulted in a total protection against 0.16 mM t-butyl hydroperoxide.

Protein N-terminal Acetylation by the NatA Complex Is Critical for Selective Mitochondrial Degradation

Mitophagy is an evolutionarily conserved autophagy pathway that selectively degrades mitochondria. Although it is well established that this degradation system contributes to mitochondrial quality and quantity control, mechanisms underlying mitophagy remain largely unknown. Here, we report that protein N-terminal acetyltransferase A (NatA), an enzymatic complex composed of the catalytic subunit Ard1 and the adaptor subunit Nat1, is crucial for mitophagy in yeast. NatA is associated with the ribosome via Nat1 and acetylates the second amino acid residues of nascent polypeptides. Mitophagy, but not bulk autophagy, is strongly suppressed in cells lacking Ard1, Nat1, or both proteins. In addition, loss of NatA enzymatic activity causes impairment of mitochondrial degradation, suggesting that protein N-terminal acetylation by NatA is important for mitophagy. Ard1 and Nat1 mutants exhibited defects in induction of Atg32, a protein essential for mitophagy, and formation of mitochondria-specific autophagosomes. Notably, overexpression of Atg32 partially recovered mitophagy in NatA-null cells, implying that this acetyltransferase participates in mitophagy at least in part via Atg32 induction. Together, our data implicate NatA-mediated protein modification as an early regulatory step crucial for efficient mitophagy.

Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein.

Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.

Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

Variable amino acid sequences in the S-loop and target binding site of vegetative actin in flowers of the Ascocenda orchid

Although plant actins have highly conserved sequences, they are classified as vegetative or reproductive actin based on genetic relationship and the role of the tissue. To characterize the 1,590 bp actin (ACT) gene cloned from the petals of the Ascocenda Princess Mikasa ‘Blue’ orchid, the encoded protein sequence of Ascocenda ACT was aligned to 31 related proteins from orchids and other plant species. The comparison of the amino acids of the actin proteins showed that Ascocenda ACT1, Cymbidium ACT, Phalaenopsis ACT1, Phalaenopsis ACT2, and Phalaenopsis ACT4 contained 13 positions different in amino acid residues in actin polypeptides. Consequently, Ascocenda ACT, Cymbidium ACT, and Phalaenopsis ACT2 were classified as vegetative actins. The classification of Ascocenda ACT was consistent with the results of qualtitative real-time polymerase chain reaction (qPCR) analysis, which demonstrated that actin is highly expressed in all vegetative tissues of the Ascocenda orchid but not in pollen. The predicted three-dimensional structure of the orchid actins revealed that most of the 13 variable residues were located outside the dynamic functional loops, with the exception of Thr16 and Pro353, which were located in the S-loop and target binding site of Ascocenda ACT, respectively. We suggest that the DNA sequence in actin may play a role in the functions of each actin isoform in a tissue-specific manner.

PH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

The pK a values and charge states of ionizable residues in polypeptides and proteins are frequently determined via NMR-monitored pH titrations. To aid the interpretation of the resulting titration data, we have measured the pH-dependent chemical shifts of nearly all the (1)H, (13)C, and (15)N nuclei in the seven common ionizable amino acids (X=Asp, Glu, His, Cys, Tyr, Lys, and Arg) within the context of a blocked tripeptide, acetyl-Gly-X-Gly-amide. Alanine amide and N-acetyl alanine were used as models of the N- and C-termini, respectively. Together, this study provides an essentially complete set of pH-dependent intra-residue and nearest-neighbor reference chemical shifts to help guide protein pK a measurements. These data should also facilitate pH-dependent corrections in algorithms used to predict the chemical shifts of random coil polypeptides. In parallel, deuterium isotope shifts for the side chain (15)N nuclei of His, Lys, and Arg in their positively-charged and neutral states were also measured. Along with previously published results for Asp, Glu, Cys, and Tyr, these deuterium isotope shifts can provide complementary experimental evidence for defining the ionization states of protein residues.

Oxidation of Methionine Residues in Polypeptide Ions Via Gas-Phase Ion/Ion Chemistry

The gas-phase oxidation of methionine residues is demonstrated here using ion/ion reactions with periodate anions. Periodate anions are observed to attach in varying degrees to all polypeptide ions irrespective of amino acid composition. Direct proton transfer yielding a charge-reduced peptide ion is also observed. In the case of methionine and, to a much lesser degree, tryptophan-containing peptide ions, collisional activation of the complex ion generated by periodate attachment yields an oxidized peptide product (i.e., [M + H + O](+)), in addition to periodic acid detachment. Detachment of periodic acid takes place exclusively for peptides that do not contain either a methionine or tryptophan side chain. In the case of methionine-containing peptides, the [M + H + O](+) product is observed at a much greater abundance than the proton transfer product (viz., [M + H](+)). Collisional activation of oxidized Met-containing peptides yields a signature loss of 64Da from the precursor and/or product ions. This unique loss corresponds to the ejection of methanesulfenic acid from the oxidized methionine side chain and is commonly used in solution-phase proteomics studies to determine the presence of oxidized methionine residues. The present work shows that periodate anions can be used to 'label' methionine residues in polypeptides in the gas phase. The selectivity of the periodate anion for the methionine side chain suggests several applications including identification and location of methionine residues in sequencing applications.

Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

AbstractThe enzyme sortase A is a ligase which catalyzes transpeptidation reactions.1, 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly5 on the peptidoglycan of bacterial cell walls by sortase A.1 The enzyme is an important anti‐virulence and anti‐infective drug target for resistant strains of Gram‐positive bacteria.2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science.3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase AΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size‐exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.

Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

The enzyme sortase A is a ligase which catalyzes transpeptidation reactions.1, 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly5 on the peptidoglycan of bacterial cell walls by sortase A.1 The enzyme is an important anti-virulence and anti-infective drug target for resistant strains of Gram-positive bacteria.2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science.3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase AΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size-exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.

Surviving a Bumpy Ride in the Oropharynx: Bacterial Pili as Nano-Seatbelts that Dissipate Mechanical Energy

Bacterial pili function in cellular adhesion, and must withstand large mechanical stresses in host environments, such as coughing and chewing. In gram positive bacteria, pili are covalently-linked polymers of single protein subunits, termed pilins. Gram positive pilins uniquely possess intramolecular isopeptide bonds that bridge the peptide backbone to form bypass force transduction pathways. In the crystal structure of Spy0128, a pilin from Streptococcus pyogenes, isopeptide bonds link the N- and C-terminal β-strands. Consequently, Spy0128 is mechanically inextensible. Here we report on the mechanical properties of two related pilins, SpaA from Corynebacterium diphtheriae and FimA from Actinomyces oris, using atomic force microscopy (AFM)-based single molecule force spectroscopy. In the crystal structures of SpaA and FimA, the isopeptide bonds do not directly link the N- and C-terminal β-strands in a single pilin domain. Instead, the isopeptide arrangement creates a ∼40 residue polypeptide loop that resembles a slackened seatbelt, which we predict is sensitive to mechanical unfolding. We find that both SpaA and FimA extend to 14 nm under mechanical force, consistent with our structure-based prediction of unfolding of the nano-seatbelt from a slackened to a taut conformation. At a loading rate of 400 nm/s, these loops unfold at forces of ∼503pN in SpaA and ∼665pN in FimA; as such, SpaA and FimA are among the most mechanically stable proteins yet reported. When the force perturbation is removed, the loops refold at a rapid rate of 29 s−1 or higher. Remarkably, the mechanical stabilities are ∼75pN weaker upon refolding, suggesting that gaining full mechanical stability requires maturation. The high mechanical stability and rapid refolding of the nano-seatbelts suggest a mechanism whereby pilin subunits, polymerized as tens-to-hundreds of repeats in pili, readily absorb and recover from mechanical shocks.

Cyclic N-Terminal Loop of Amylin Forms Non Amyloid Fibers

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1–8) forms extremely long and stable non-β-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.

Isolation of an alcohol dehydrogenase cDNA from and characterization of its expression in chrysanthemum under waterlogging

A PCR strategy was used to isolate a full-length CgADH (alcohol dehydrogenase) cDNA from chrysanthemum. The gene putatively encodes a 378 residue polypeptides, which shares 95% homology with tomato alcohol dehydrogenase class III. Endogenous ethylene generated in waterlogged Chrysanthemum zawadskii was enhanced by exogenous ethylene but decreased by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action. In waterlogged roots, the transcription of the gene encoding alcohol dehydrogenase (ADH, EC 1.1.1.1) increased rapidly but transiently, peaking at 7.5 fold the non-waterlogged level after 2h of stress. Waterlogging elevated ADH activity after a prolonged episode of stress. The exogenous supply of 40μLL−1 ethylene suppressed the production of ethanol, while that of 4μLL−1 1-MCP enhanced it. Ethylene appeared to suppress an acceleration of both CgADH expression and fermentation, and alleviates ethanolic fermentation probably through by as a signal to acceleration of waterlogging-induced aerenchyma formation. This supports the previously observed phenomenon that the expression level of ADH gene is regulated by the local level of physiologically active ethylene. The relevance of the CgADH gene in relation to chrysanthemum waterlogging was discussed as well.

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31mM and 132μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30°C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.

Molecular characterization and functional analysis of the Nep1-like protein-encoding gene from Phytophthora capsici

Phytophthora capsici is an aggressive plant pathogen that affects solanaceous and cucurbitaceous hosts. Nep1-like proteins (NLPs) are a group of effectors found particularly in oomycetes and considered important virulence factors. We identified an NLP gene (phcnlp1) from the highly virulent P. capsici strain Phyc12 that had an encoded polypeptide of 476-amino acid residues and a predicted molecular mass of 51.75 kDa. We performed quantitative reverse transcription-polymerase chain reaction to detect the expression pattern of phcnlp1 during various phases of interaction with the host plant, and the results showed that phcnlp1 was increasingly expressed during symptom development after P. capsici infection of pepper leaves. We also confirmed that phcnlp1 caused significant necrosis on tobacco plants when expressed based on potato virus agroinfection. All results indicated that phcnlp1 belongs to the NLP gene family and is important for the pathogenesis of P. capsici in its hosts.

Modulating Amyloid Aggregation by Incorporation of Fluorinated Phenylalanine Derivatives in the Central Hydrophobic Cluster of Aβ10-35

Aromatic amino acids have been suspected to drive the aggregation mechanism in amyloidogenic peptides by virtue of their hydrophobicity and aromaticity. Alzheimer's disease in particular is associated with the aggregation of a 39-42 residue polypeptide (Amyloid β, Aβ) and its subsequent deposition into amyloid plaques. The aromatic phenylalanine residues (Phe19 and Phe20) in the central hydrophobic cluster (CHC) of the peptide have been suspected to play a significant role in such aggregation. In the present study the amyloid β 10-35 (Aβ10-35) fragment has been used as a model system, where the CHC has been perturbed through the introduction of non-natural amino acids. We have prepared variants where Phe19 and Phe20 have been systematically replaced by pentafluorophenylalanine (F5-Phe) and their affect on amyloid aggregation and kinetics has been studied by biophysical techniques including circular dichroism (CD), fluorescence, UV resonance Raman (UVRR) spectroscopy and chemometrics. While Phe→F5-Phe mutations have been found to enhance conformational stability in Aβ16-22 (Senguen et al. Mol. BioSyst., 2011, 7, p486) by virtue of enhanced hydrophobicity, unfavorable steric interactions brought about by such substitution can also lead to destabilization in some systems (Cornilescu et al., Prot.Sc., 2007, 16, p14). Our experimental studies show that aggregation in both the wild type (wT) Aβ10-35 and the Phe 19→F5-Phe mutant proceeds via a two step conformational transition pathway (wTAβ10-35: T1= 9.18 ±1.4 hrs, T2= 9.79 ±0.44 days, F5-Phe19: T1= 10.72 ± 2.1 hrs, T2= 7.6 ± 0.47 days). However the Phe 20→F5-Phe mutants fail to aggregate into β-sheets as confirmed by both CD and fluorescence studies. Deep UVRR studies have also been performed to probe the fibrillation process and monitor the structural changes brought about by the F5-Phe substitution and their effect on amyloid aggregation.