Improved sonolytic hydrolysis of peptides in aqueous solution with addition of 1,4-benzenedithiol

Described here is the sonolytic hydrolysis of peptides achieved by treatment of aqueous solution to which the radical scavenger 1,4-benzenedithiol (1,4-BDT), which has hydrogen donating ability, has been added. Mass spectrometric analysis of the products of sonolytic hydrolysis gave information about amino acid sequence of the peptides without any byproducts. The additive 1,4-BDT improves the sonolytic hydrolysis of peptides in terms of the rate of hydrolysis reaction and the amount of additive required when compared to catechol, a previously reported additive. The sonolytic hydrolysis of peptides differs from both acid hydrolysis and hydrogen atom-induced dissociation named matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD), in characteristics. We propose a mechanistic reaction for the sonolytic hydrolysis of peptides, based on the mechanisms of both acid hydrolysis and MALDI-ISD processes. The sonolytic hydrolysis of peptides upon addition of hydrogen donating radical scavengers can be rationalized via the attachment of a hydrogen atom to the carbonyl oxygen with subsequent hydrolysis.     

Comparison of CID, ETD and metastable atom-activated dissociation (MAD) of doubly and triply charged phosphorylated tau peptides

The fragmentation behavior of the 2+ and 3+ charge states of eleven different phosphorylated tau peptides was studied using collision-induced dissociation (CID), electron transfer dissociation (ETD) and metastable atom-activated dissociation (MAD). The synthetic peptides studied contain up to two known phosphorylation sites on serine or threonine residues, at least two basic residues, and between four and eight potential sites of phosphorylation. CID produced mainly b-/y-type ions with abundant neutral losses of the phosphorylation modification. ETD produced c-/z-type ions in highest abundance but also showed numerous y-type ions at a frequency about 50% that of the z-type ions. The major peaks observed in the ETD spectra correspond to the charge-reduced product ions and small neutral losses from the charge-reduced peaks. ETD of the 2+ charge state of each peptide generally produced fewer backbone cleavages than the 3+ charge state, consistent with previous reports. Regardless of charge state, MAD achieved more extensive backbone cleavage than CID or ETD, while retaining the modification(s) in most cases. In all but one case, unambiguous modification site determination was achieved with MAD. MAD produced 15-20% better sequence coverage than CID and ETD for both the 2+ and 3+ charge states and very different fragmentation products indicating that the mechanism of fragmentation in MAD is unique and complementary to CID and ETD.     

Strong UV absorption and visible luminescence in ytterbium-doped aluminosilicate glass under UV excitation

A broad visible luminescence band and characteristic IR luminescence of Yb(3+) ions are observed under UV excitation in ytterbium-doped aluminosilicate glass. Samples made under both oxidizing and reducing conditions are analyzed. A strong charge-transfer absorption band in the UV range is observed for glass samples containing ytterbium. Additional absorption bands are observed for the sample made under reducing conditions, which are associated with f-d transitions of divalent ytterbium. The visible luminescence band is attributed to 5d-4f emission from Yb(2+) ions, and the IR luminescence is concluded to originate from a relaxed charge-transfer transition. The findings are important to explain induced optical losses (photodarkening) in high-power fiber lasers.     

Structural characterization and angiotensin-converting enzyme (ACE) inhibitory mechanism of Stropharia rugosoannulata mushroom peptides prepared by ultrasound

To reveal the structural characteristics and angiotensin-converting enzyme (ACE) inhibition mechanism of Stropharia rugosoannulata mushroom peptides prepared by multifrequency ultrasound, the peptide distribution, amino acid sequence composition characteristics, formation pathway, and ACE inhibition mechanism of S. rugosoannulata mushroom peptides were studied. It was found that the peptides in S. rugosoannulata mushroom samples treated by multifrequency ultrasound (probe ultrasound and bath ultrasound mode) were mainly octapeptides, nonapeptides, and decapeptides. Hydrophobic amino acids were the primary amino acids in the peptides prepared by ultrasound, and the amino acid dissociation of the peptide bonds at the C-terminal under the action of ultrasound was performed mainly to produce hydrophobic amino acids. Pro and Val (PV), Arg and Pro (RP), Pro and Leu (PL), and Asp (D) combined with hydrophobic amino acids were the characteristic amino acid sequence basis of the active peptides of the S. rugosoannulata mushroom. The docking results of active peptides and ACE showed that hydrogen bond interaction remained the primary mode of interaction between ACE and peptides prepared by ultrasound. The peptides can bind to the amino acid residues in the ACE active pocket, zinc ions, or key amino acids in the domain, and this results in inhibition of ACE activity. Cation–pi interactions also played an important role in the binding of mushroom peptides to ACE. This study explains the structural characteristics and ACE inhibition mechanism used by S. rugosoannulata mushroom peptides prepared by ultrasound, and it will provide a reference for the development and application of S. rugosoannulata mushroom peptides.     

西南印度洋中脊热液区海底玄武岩元素地球化学原位分析

对西南印度洋中脊壬液区海底新鲜和蚀变玄武岩样品进行了原位元素地球化学分析,发现其中的蚀变矿物主要为绿鳞石。电子探针及能谱线扫分析显示杏仁状绿鳞石从核部到边部具有K含量降低而Na含量有所升高的特点,指示边部绿鳞石转化为皂石。绿鳞石为低温(<50 ℃)的氧化条件下的蚀变矿物,而皂石则在较低水/岩值、还原性更强的条件下形成,转化过程暗示了蚀变环境由氧化环境转化为还原环境。以电子探针为内标,利用LA-ICP-MS对蚀变矿物做原位的稀土元素分析,大部分结果均有Eu正异常的特点,指示了蚀变流体从氧化性向还原性转变,并且还原性流体对早期蚀变矿物进行了叠加改造。而蚀变区和未蚀变区的基质长石原位稀土元素对比研究则表明后期还原性热液蚀变流体对稀土元素进行了淋滤,从而导致了蚀变区稀土总量下降且出现Eu正异常的特点。由此可见,热液系统中淋滤作用具有重要意义。     

N-磷酰化叉肽结构与NMR波谱

测定了N-磷酰化叉肽(2-5)在溶液中的NMR谱.发现它们具有非对映异位,分子无规卷曲及氢键相互作用.波谱分析表明,磷酰基的参与,主要影响其结合部位的氨基酸.由于肽平面的生成及分子内、分子间氢键影响的改变,致使叉肽中支链的Dipp-Ala磷酰基与β位羰基的相对位置发生变化.磷酰基与N-C_α基团仍处于反式位置,这可能是该氨基酸侧链无极性基团的缘故.     

Gold nanoparticle-enhanced secondary ion mass spectrometry and its bio-applications

Enhancement of signals in time-of-flight secondary ion mass spectrometry (ToF-SIMS) studies is necessary to many biological applications. We have developed an efficient method of enhancing the signals of secondary ions from peptides using gold nanoparticles (AuNPs) attached to a well-controlled surface such as self-assembled monolayers (SAMs). AuNPs function as both signal enhancers and effective binding sites for peptides, which allow the high signal intensity from the peptides to produce well-contrasted ToF-SIMS images of peptides that are micropatterned on the surface of the AuNPs. For application, this AuNP-enhanced SIMS (NE-SIMS) provided the basis for the spectrum and images to assay protein kinases and their inhibitors. Phosphorylation efficiency and inhibitor effect were quantified by detecting mass change of the peptide substrates by kinase reaction. Maximum efficiency of phosphorylation was achieved from cysteine-tethered peptides with a spacer linker, indicating that accessibility of kinase was dependent on the surface orientation and length of the peptide substrate on the three-dimensional AuNPs. The activities of other enzymes such as phosphatase and protease could also be assayed using NE-SIMS. Our study shows that NE-SIMS can be applied as a useful tool for enzyme assay in biochip surfaces.     

Interactions between oligopeptides and oxidised titanium surfaces detected by vibrational spectroscopy

Five alternating polar/hydrophobic oligopeptides derived from EAK 16 (AEAEAKAKAEAEAKAK) were examined in comparison with EAK 16 (peptide 1) both after solubilisation/lyophilisation and deposition on oxidised titanium surfaces. The peptides were synthesised for their possible use as biomimetic materials due to their self‐assembling properties and the presence, in one of them, of the arginine‐glycine‐aspartic (RGD) sequence, an active modulator of cell adhesion. Infrared (IR) and Raman spectroscopies were used to investigate the influence of the amino acid substitution on the self‐assembling properties of the peptides under both experimental conditions. In the lyophilised peptides, β‐sheet was the prevailing conformation (65–69%) as in EAK 16, irrespective of acid substitution (E→D, peptide 2), basic substitution (K→O, peptide 3), hydrophobic spacer substitution (A→Abu, peptide 4 and A→Y, peptide 5) and RGD insertion (peptide 6). After deposition on oxidised titanium, the main conformation remained β‐sheet. The side‐chain shortening of the acidic amino acid residue (peptide 2) or the insertion of a rigid and bulky residue such as Y (peptide 5) decreased the self‐assembling ability more than the side‐chain shortening of the basic amino acid residue (peptide 3) or the insertion of the RGD head (peptide 6). The interaction with the oxidised titanium surface was mainly due to carboxylate groups with a bidentate bridging coordination and C  O peptidic groups. Copyright © 2010 John Wiley & Sons, Ltd.     

Isosteres of peptides: boron analogs as dipolar forms of α‐amino acids – a theoretical study

Modification of peptides to produce peptidomimetics is of great interest, with the aim of designing potent, selective, and metabolically stable analogs having certain conformational properties. Organoboranes have been reported in the literature with a wide range of therapeutic applications. One of the therapeutically important class of molecules is amine‐carboxyboranes derived from amino acids by replacement of the Cα atom of an amino acid/peptide by boron. The conformational preferences of these peptides, with respect to backbone ω, ?, and ψ torsion angles, have been investigated by theoretical calculations. The amide bond in these molecules has the same geometry in the ground and transition states as the natural peptides. However, the boron isosteres of glycine and alanine peptides are less structured than their natural derivatives, but are distinguished by structures with a positive value for the ? angle, which is normally disfavored for natural peptides. This property could be used to build peptides with a geometry not usually seen in natural peptides. Copyright © 2007 John Wiley & Sons, Ltd.     

Vibrational spectroscopy of self‐assembling aromatic peptide derivates

Peptides can assemble to supramolecular structures, of which fibers are of special biochemical and medical relevance. We employed Raman and infrared spectroscopy to elucidate the chemical integrity of fibers built from peptides and peptide derivates that contain the aromatic side groups fluorenyl and phenyl. Because the observed spectra compare very well with simulation results of the respective single molecules in vacuum, we were able to assign all observed vibrations. We found the main differences between solid phase and single molecule for O‐H and N‐H stretching and bending vibrations, owing to hydrogen bonding in solids. The fluorenyl and phenyl residues cause π‐stacking of the molecules, which barely manifests in the spectra, but clearly in the structures. Whereas hydrogen bonds provide the principal stability of the fiber backbone, aromatic π‐stacking supports the assembly to fibers, especially when electrospinning assists the molecular alignment. Copyright © 2012 John Wiley & Sons, Ltd.     

Study on the relationship between structure and taste activity of the umami peptide of Stropharia rugosoannulata prepared by ultrasound

Through virtual screening, electronic tongue verification, and molecular docking technology, the structure-taste activity relationship of 47 kinds of umami peptides (octapeptide - undecapeptide) from Stropharia rugosoannulata prepared by simultaneous ultrasonic-assisted directional enzymatic hydrolysis was analyzed. The umami peptides of S.rugosoannulata can form hydrogen bond interaction and electrostatic interaction with umami receptors T1R1/T1R3. The amino acid residues at the peptides' N-terminal and C-terminal play a vital role in binding with the receptors to form a stable complex. D, E, and R are the primary amino acids in the peptides that easily bind to T1R1/T1R3. The basic amino acid in the peptides is more easily bound to T1R1, and the acidic amino acid is more easily bound to T1R3. The active amino acid sites of the receptors to which the peptides bind account for 42%−65% of the total active amino acid residues in the receptors. ASP147 and ASP219 are the critical amino acid residues for T1R1 to recognize the umami peptides, and ARG64, GLU45, and GLU48 are the critical amino acid residues for T1R3 to recognize the umami peptides. The increase in the variety and quantity of umami peptides is the main reason for improving the umami taste of the substrate prepared by synchronous ultrasound-assisted directional enzymatic hydrolysis. This study provides a theoretical basis for understanding simultaneous ultrasound-assisted directional enzymatic hydrolysis for preparing umami peptides from S.rugosoannulata, enhancing the flavor of umami, and the relationship between peptide structure and taste activity.     

A novel surface cleaning method for chemical removal of fouling lead layer from chromium surfaces

Most products especially metallic surfaces require cleaning treatment to remove surface contaminations that remain after processing or usage. Lead fouling is a general problem which arises from lead fouling on the chromium surfaces of bores and other interior parts of systems which have interaction with metallic lead in high temperatures and pressures. In this study, a novel chemical solution was introduced as a cleaner reagent for removing metallic lead pollution, as a fouling metal, from chromium surfaces. The cleaner aqueous solution contains hydrogen peroxide (H₂O₂) as oxidizing agent of lead layer on the chromium surface and acetic acid (CH₃COOH) as chelating agent of lead ions. The effect of some experimental parameters such as acetic acid concentration, hydrogen peroxide concentration and temperature of the cleaner solution during the operation on the efficiency of lead cleaning procedure was investigated. The results of scanning electron microscopy (SEM) showed that using this procedure, the lead pollution layer could be completely removed from real chromium surfaces without corrosion of the original surface. Finally, the optimum conditions for the complete and fast removing of lead pollution layer from chromium surfaces were proposed. The experimental results showed that at the optimum condition (acetic acid concentration 28% (V/V), hydrogen peroxide 8% (V/V) and temperature 35 °C), only 15-min time is needed for complete removal of 3 g fouling lead from a chromium surface.     

Modulation of intra- and inter-sheet interactions in short peptide self-assembly by acetonitrile in aqueous solution

Besides our previous experimental discovery(Zhao Y R, et al. 2015 Langmuir, 31, 12975) that acetonitrile(ACN)can tune the morphological features of nanostructures self-assembled by short peptides KIIIIK(KI4K) in aqueous solution,further experiments reported in this work demonstrate that ACN can also tune the mass of the self-assembled nanostructures.To understand the microscopic mechanism how ACN molecules interfere peptide self-assembly process, we conducted a series of molecular dynamics simulations on a monomer, a cross-β sheet structure, and a proto-fibril of KI4 K in pure water, pure ACN, and ACN-water mixtures, respectively. The simulation results indicate that ACN enhances the intra-sheet interaction dominated by the hydrogen bonding(H-bonding) interactions between peptide backbones, but weakens the inter-sheet interaction dominated by the interactions between hydrophobic side chains. Through analyzing the correlations between different groups of solvent and peptides and the solvent behaviors around the proto-fibril, we have found that both the polar and nonpolar groups of ACN play significant roles in causing the opposite effects on intermolecular interactions among peptides. The weaker correlation of the polar group of ACN than water molecule with the peptide backbone enhances H-bonding interactions between peptides in the proto-fibril. The stronger correlation of the nonpolar group of ACN than water molecule with the peptide side chain leads to the accumulation of ACN molecules around the proto-fibril with their hydrophilic groups exposed to water, which in turn allows more water molecules close to the proto-fibril surface and weakens the inter-sheet interactions. The two opposite effects caused by ACN form a microscopic mechanism clearly explaining our experimental observations.     

Cysteine‐linked aromatic nitriles as UV resonance Raman probes of protein structure

Nitriles introduced into peptides and proteins can serve as useful vibrational spectroscopic probes, because the nitrile C ≡ N stretch is well isolated from backbone and sidechain vibrational bands. Aromatic nitriles offer large νC ≡ N absorption intensity in infrared spectra and resonance enhancement in Raman spectra with ultraviolet excitation. We report the ultraviolet resonance Raman spectra of cyanophenylalanine attached to cysteine, through linkage reactions that are applicable to cysteine residues in proteins. Excitation profiles are reported, and the νC ≡ N detection limit is estimated to be 5 µ m . The band position is sensitive to solvent polarity and especially to strong H‐bonding. The derivatization of mastoparan X peptide at introduced cysteine residues demonstrated the effectiveness of a cyanophenylcysteine probe in reporting the lowered environmental polarity when the peptide was incorporated into liposomes. For an asymmetrical cyanophenyl derivative, 2‐CBCys, the intensity ratio of asymmetric and symmetric ring modes, ν8_b and ν8_a, was found to respond to solvent polarity and not to H‐bonding. Copyright © 2012 John Wiley & Sons, Ltd.     

Mass Spectrometric (FAB) Study of Aspartic Acid Side Reactions in Peptide Synthesis

Abstract

We have been interested in the synthesis of branched peptides, by grafting an amine, eg histamine, to the carboxylic group of aspartic acid side chain of Boc-β Ala-Trp-Met-Asp-Phe-NH₂ also called Pentagastrin (or α-PG). Depending on the coupling conditions used, the main product obtained might be either the wanted derivative or a side-product identified a6 amino-succinyl-pentagastrin (or ASC-PG). Acid or base treatment of this product cleaved the amino-succinyl ring and yielded either α-PG or/and (β-Aspartyl)⁴-PC or β-PG. Amino-succinylation being a general problem in peptide synthesis of aspartyl residue containing peptides, synthesis of α-PG, β-PG, Asc-PG and their corresponding C-terminal dipeptide amides were performed to be analyzed by mass spectroscopy.

FAB mass spectra (PI in glycerol) of the free peptides, as well as of their N-Boc derivatives have been recorded and compared to the mono, di, tri and tetra peptide derivatives. The pseudo-molecular ions of Boc-peptides are usually not observed. However, the intense M+1-Boc (? 100 u) ions are present.

Several sequence ions have been identified and compared to the simulation of their spectra according to six fragmentation routes.

The identification of α, β or succinimide structures from FAB spectra has been proposed.     

Covalent and non-covalent binding in the ion/ion charge inversion of peptide cations with benzene-disulfonic acid anions

Protonated angiotensin II and protonated leucine enkephalin-based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4-formyl-1,3-benzenedisulfonic acid (FBDSA) and 1,3-benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M + FBDSA-H](-) via collisional-induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e. no covalent bond formation). A series of studies with model systems confirmed that strong non-covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically bound complexes. The results reported here shed new light on the nature of non-covalent interactions in gas phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications.     

Representing environment-induced helix-coil transitions in a coarse grained peptide model

Coarse grained (CG) models are widely used in studying peptide self-assembly and nanostructure formation. One of the recurrent challenges in CG modeling is the problem of limited transferability, for example to different thermodynamic state points and system compositions. Understanding transferability is generally a prerequisite to knowing for which problems a model can be reliably used and predictive. For peptides, one crucial transferability question is whether a model reproduces the molecule's conformational response to a change in its molecular environment. This is of particular importance since CG peptide models often have to resort to auxiliary interactions that aid secondary structure formation. Such interactions take care of properties of the real system that are per se lost in the coarse graining process such as dihedral-angle correlations along the backbone or backbone hydrogen bonding. These auxiliary interactions may then easily overstabilize certain conformational propensities and therefore destroy the ability of the model to respond to stimuli and environment changes, i.e. they impede transferability. In the present paper we have investigated a short peptide with amphiphilic EALA repeats which undergoes conformational transitions between a disordered and a helical state upon a change in pH value or due to the presence of a soft apolar/polar interface. We designed a base CG peptide model that does not carry a specific (backbone) bias towards a secondary structure. This base model was combined with two typical approaches of ensuring secondary structure formation, namely a C_α-C_α-C_α-C_α pseudodihedral angle potential or a virtual site interaction that mimics hydrogen bonding. We have investigated the ability of the two resulting CG models to represent the environment-induced conformational changes in the helix-coil equilibrium of EALA. We show that with both approaches a CG peptide model can be obtained that is environment-transferable and that correctly represents the peptide's conformational response to different stimuli compared to atomistic reference simulations. The two types of auxiliary interactions lead to different kinetic behavior as well as to different structural properties for fully formed helices and folding intermediates, and we discuss the advantages and disadvantages of these approaches.     

Ultraviolet resonance Raman spectroscopy of a β‐sheet peptide: a model for membrane protein folding

The partitioning of a hydrophobic hexapeptide, N‐acetyl‐tryptophan‐pentaleucine (AcWL5), into self‐associated β‐sheets within a vesicle membrane was studied as a model for integral membrane protein folding and insertion via vibrational and electronic spectroscopy. Ultraviolet resonance Raman spectroscopy allows selective examination of the structures of amino acid side chains and the peptide backbone and provides information about local environment and molecular conformation. The secondary structure of AcWL5 within a vesicle membrane was investigated using 207.5‐nm excitation and found to consist of β‐sheets, in agreement with previous studies. The β‐sheet peptide shows enhanced Raman scattering cross‐sections for all amide modes as well as extensive hydrogen‐bonding networks. Tryptophan vibrational structure was probed using 230‐nm excitation. Increases in Raman cross‐sections of tryptophan modes W1, W3, W7, W10, W16, W17, and W18 of membrane‐incorporated AcWL5 are primarily attributed to greater resonance enhancement with the B_b electronic transition. The W17 mode, however, undergoes a much greater enhancement than is expected for a simple resonance effect, and this observation is discussed in terms of hydrogen bonding of the indole ring in a hydrophobic environment. The observed tryptophan mode frequencies and intensities overall support a hydrophobic environment for the indole ring within a vesicle, and these results have implications for the location of tryptophan in membrane protein systems. Copyright © 2009 John Wiley & Sons, Ltd.     

Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels

A highly sensitive solid state (19)F-NMR strategy is described to determine the orientation and dynamics of membrane-associated peptides from specific fluorine labels. Several analogues of the antimicrobial peptide PGLa were synthesized with the non-natural amino acid 4-trifluoromethyl-phenylglycine (CF(3)-Phg) at different positions throughout the alpha-helical peptide chain. A simple 1-pulse (19)F experiment allows the simultaneous measurement of both the anisotropic chemical shift and the homonuclear dipolar coupling within the rotating CF(3)-group in a macroscopically oriented membrane sample. The value and sign of the dipolar splitting determines the tilt of the CF(3)-rotational axis, which is rigidly attached to the peptide backbone, with respect to the external magnetic field direction. Using four CF(3)-labeled peptide analogues (with L-CF(3)-Phg at Ile9, Ala10, Ile13, and Ala14) we confirmed that PGLa is aligned at the surface of lipid membranes with its helix axis perpendicular to the bilayer normal at a peptide:lipid ratio of 1:200. We also determined the azimuthal rotation angle of the helix, which agrees well with the orientation expected from its amphiphilic character. Peptide analogues with a D-CF(3)-Phg label resulting from racemization of the amino acid during synthesis were separately collected by HPLC. Their spectra provide additional information about the PGLa structure and orientation but allow only to discriminate qualitatively between multiple solutions. The structural and functional characterization of the individual CF(3)-labeled peptides by circular dichroism and antimicrobial assays showed only small effects for our four substitutions on the hydrophobic face of the helix, but a significant disturbance was observed in a fifth analogue where Ala8 on the hydrophilic face had been replaced. Even though the hydrophobic CF(3)-Phg side chain cannot be utilized in all positions, it allows highly sensitive NMR measurements over a wide range of experimental conditions and dynamic regimes of the peptide.     

Peptide backbone-copper ring structure: A molecular insight into copper-induced amyloid toxicity

Copper ions can promote amyloid diseases that are associated with amyloid peptides, such as type 2 diabetes (T2D), Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). However, the underlying molecular mechanism remains obscure. Here we present that Cu²⁺ is able to specifically bind to the backbone of T2D-related human islet amyloid polypeptide (hIAPP) by forming a ring structure, which causes the reduction of Cu²⁺ to Cu⁺ to produce reactive oxygen species (ROS) and the modulation of hIAPP aggregation. Nuclear magnetic resonance spectroscopy showed that Cu²⁺ bound to the backbone of a turn region, His18—Ser21, which is critical for hIAPP aggregation. Ab initio calculations and x-ray absorption fine structure analyses revealed that Cu²⁺ simultaneously bound with both the amide nitrogen and carbonyl oxygen on the peptide backbone, resulting in a ring structure, and causing the reduction of Cu²⁺ to Cu⁺ to form a hIAPP-Cu⁺ complex. 2',7'-dichlorodihydrofluorescin diacetate fluorescence measurements further indicated that this complex led to enhanced ROS levels in rat insulinoma cells. Additionally, thioflavin T fluorescence and atomic force microscopy measurements denoted that the backbone-Cu ring structure largely modulated hIAPP aggregation, including the inhibition of hIAPP fibrillation and the promotion of peptide oligomerization. These findings shed new light on the molecular mechanism of Cu²⁺-induced amyloid toxicity involving both the enhancement of ROS and the modulation of hIAPP aggregation.