Antiparallel Β-sheet Structure Research Articles

Disulfide-Driven Charge and Hydrophobicity Rearrangement of Remodeled Membrane Proteins toward Amyloid-Type Aggregation.

As a common pathological hallmark, protein aggregation into amyloids is a highly complicated phenomenon, attracting extensive research interest for elucidating its structural details and formation mechanisms. Membrane deposition and disulfide-driven protein misfolding play critical roles in amyloid-type aggregation, yet the underlying molecular process remains unclear. Here, we employed sum frequency generation (SFG) vibrational spectroscopy to comprehensively investigate the remodeling process of lysozyme, as the model protein, into amyloid-type aggregates at the cell membrane interface. It was discovered that disulfide reduction concurrently induced the transition of membrane-bound lysozyme from predominantly α-helical to antiparallel β-sheet structures, under a mode switch of membrane interaction from electrostatic to hydrophobic, and subsequent oligomeric aggregation. These findings shed light on the systematic understanding of dynamic molecular mechanisms underlying membrane-interactive amyloid oligomer formation.

Parallel β-Sheet Structure and Structural Heterogeneity Detected within Q11 Self-Assembling Peptide Nanofibers.

Q11 peptide nanofibers are used as a biomaterial for applications such as antigen presentation and tissue engineering, yet detailed knowledge of molecular-level structure has not been reported. The Q11 peptide sequence was designed using heuristics-based patterning of hydrophobic and polar amino acids with oppositely charged amino acids placed at opposite ends of the sequence to promote antiparallel β-sheet formation. In this work, we employed solid-state nuclear magnetic resonance spectroscopy (NMR) to evaluate whether the molecular organization within Q11 self-assembled peptide nanofibers is consistent with the expectations of the peptide designers. We discovered that Q11 forms a distribution of molecular structures. NMR data from two-dimensional (2D) 13C-13C dipolar-assisted rotational resonance indicate that the K3 and E9 residues between Q11 β-strands are spatially proximate (within ∼0.6 nm). Frequency-selective rotational echo double resonance (fsREDOR) on K3 Nζ and E9 Cδ-labeled sites showed that approximately 9% of the sites are close enough for salt bridge formation to occur. Surprisingly, dipolar recoupling measurements revealed that Q11 peptides do not assemble into antiparallel β-sheets as expected, and structural analysis using Fourier-transform infrared spectroscopy and 2D NMR alone can be misleading. 13C PITHIRDS-CT dipolar recoupling measurements showed that the most abundant structure consists of parallel β-sheets, in contrast to the expected antiparallel β-sheet structure. Structural heterogeneity was detected from 15N{13C} REDOR measurements, with approximately 22% of β-strands having antiparallel nearest neighbors. We cannot propose a complete structural model of Q11 nanofibers because of the complexity involved when examining structurally heterogeneous samples using NMR. Altogether, our results show that while heuristics-based patterning is effective in promoting β-sheet formation, designing a peptide sequence to form a targeted β-strand arrangement remains challenging.

Bombyx mori Silk Fibroin and Model Peptides Incorporating Arg-Gly-Asp Motifs and Their Application in Wound Dressings.

Skin plays an important role in protecting the human body from the environment, dehydration, and infection. Burns, wounds, and disease cause the skin to lose its role, but tissue-engineered skin substitutes offer the opportunity to restore skin loss. Silk fibroin from Bombyx mori (SF) has proven to be an excellent wound dressing material. In this study, we aim to develop an excellent wound dressing material by introducing three-residue sequence Arg-Gly-Asp (RGD), which is the most well-known adhesion site of fibronectin, in the films of SF and the model peptide. Its usefulness as a wound dressing material was evaluated both in vitro and in vivo. First, we showed that the flexible structures of the RGD sequence are still maintained in SF with a rigid antiparallel β-sheet structure using NMR in association with excellent wound dressings of SF containing RGD. Then, in in vitro experiments, two types of normal cells derived from human skin, normal human neonatal epidermal keratinocytes and normal human neonatal dermal fibroblasts, were used to evaluate the cell adhesion. On the other hand, in in vivo experiments, the study was conducted using a rat model of a whole skin layer defect wound. The results showed that the high-functionalized SF developed here has the potential to play a significant role in the field of wound dressings.

Intermediate Antiparallel β Structure in Amyloid β Plaques Revealed by Infrared Spectroscopic Imaging.

Aggregation of amyloid β (Aβ) peptides into extracellular plaques is a hallmark of the molecular pathology of Alzheimer's disease (AD). Amyloid aggregates have been extensively studied in vitro, and it is well-known that mature amyloid fibrils contain an ordered parallel β structure. The structural evolution from unaggregated peptide to fibrils can be mediated through intermediate structures that deviate significantly from mature fibrils, such as antiparallel β-sheets. However, it is currently unknown if these intermediate structures exist in plaques, which limits the translation of findings from in vitro structural characterizations of amyloid aggregates to AD. This arises from the inability to extend common structural biology techniques to ex vivo tissue measurements. Here we report the use of infrared (IR) imaging, wherein we can spatially localize plaques and probe their protein structural distributions with the molecular sensitivity of IR spectroscopy. Analyzing individual plaques in AD tissues, we demonstrate that fibrillar amyloid plaques exhibit antiparallel β-sheet signatures, thus providing a direct connection between in vitro structures and amyloid aggregates in the AD brain. We further validate results with IR imaging of in vitro aggregates and show that the antiparallel β-sheet structure is a distinct structural facet of amyloid fibrils.

Controlling 1D Nanostructures and Handedness by Polar Residue Chirality of Amphiphilic Peptides.

Peptide assemblies are promising nanomaterials, with their properties and technological applications being highly hinged on their supramolecular architectures. Here, how changing the chirality of the terminal charged residues of an amphiphilic hexapeptide sequence Ac-I4 K2 -NH2 gives rise to distinct nanostructures and supramolecular handedness is reported. Microscopic imaging and neutron scattering measurements show thin nanofibrils, thick nanofibrils, and wide nanotubes self-assembled from four stereoisomers. Spectroscopic and solid-state nuclear magnetic resonance (NMR) analyses reveal that these isomeric peptides adopt similar anti-parallel β-sheet secondary structures. Further theoretical calculations demonstrate that the chiral alterations of the two C-terminal lysine residues cause the formation of diverse single β-strand conformations, and the final self-assembled nanostructures and handedness are determined by the twisting direction and degree of single β-strands. This work not only lays a useful foundation for the fabrication of diverse peptide nanostructures by manipulating the chirality of specific residues but also provides a framework for predicting the supramolecular structures and handedness of peptide assemblies from single molecule conformations.

Atomic Insight on Inhibition of Fibrillization of Dipeptides by Replacement of Phenylalanine with Tryptophan.

Tryptophan (Trp) conjugates destabilize amyloid fibrils responsible for amyloidoses. However, the mechanism of such destabilization is obscure. Herein the self-assembly of four synthesized Trp-containing dipeptides Boc-xxx-Trp-OMe (xxx: Val, Leu, Ile, and Phe) has been investigated and compared with the existing report on their Phe congeners. Two among them are the C-terminal tryptophan analogs of Boc-Val-Phe-OMe (VF, Aβ18-19) and Boc-Phe-Phe-OMe (FF, Aβ19-20), part of the central hydrophobic region of amyloid-β (Aβ1-42). While Boc-Val-Trp-OMe (VW), Boc-Leu-Trp-OMe (LW), Boc-Ile-Trp-OMe (IW), and Boc-Phe-Trp-OMe (FW) displayed a spherical morphology in FESEM and AFM images, the corresponding phenylalanine-containing dipeptides displayed various fibrous structures. Single-crystal X-ray diffraction (SC-XRD) indicated that peptides VW and IW exhibited structures containing parallel β-sheet, cross-β-structure, sheet-like layer structure, and helical arrangement in the solid state. Interestingly, peptide FW displayed inverse γ-turn conformation (similar to open-turn structure), antiparallel β-sheet structure, columnar structure, supramolecular nanozipper structure, sheet-like layer arrangement, and helical architecture in the solid state. The open-turn conformation and nanozipper structure formation by FW may be the first example of a dipeptide that forms such structures. The minute but consistent differences in molecular packing at the atomic level between Trp and Phe congeners may be responsible for their remarkably different supramolecular structure generation. This molecular-level structural analysis may be helpful for the de novo design of peptide nanostructures and therapeutics. Similar studies by the Debasish Haldar group are reported, but they investigated the inhibition of fibrillization of dipeptides by tyrosine and interactions are expectedly different.

Flow-Induced Protein Chain Deformation, Segmental Orientation, and Phase Separation in Native Silk Feedstock.

The ability of many arthropods to spin silk and its many uses bear testament to its importance in Nature. Despite over a century of research, however, the spinning process is still not fully understood. While it is widely accepted that flow and chain alignment may be involved, the link to protein gelation remains obscure. Using combinations of rheology, polarized light imaging, and infrared spectroscopy to probe different length scales, this work explored flow-induced gelation of native silk feedstock from Bombyx mori larvae. Protein chain deformation, orientation, and microphase separation were observed, culminating in the formation of antiparallel β-sheet structures while the work rate during flow appeared as an important criterion. Moreover, infrared spectroscopy provided direct observations suggesting a loss of protein hydration during flow-induced gelation of fibroin in native silk feedstock, which is consistent with recently reported hypotheses.

Exploration of N-Arylation of Backbone Amides as a Novel Tool for Conformational Modification in Peptides.

A set of 15 cyclic-hexaalanine and 10 cyclic-pentaalanine peptides containing one or two backbone N-aryl amide bonds were synthesized by following a combination of solution-phase and solid-phase peptide synthesis. NMR-based conformation studies of these N-aryl cyclic-hexaalanine peptides revealed five distinct template conformations with an antiparallel β-sheet structure; for N-aryl cyclic-pentaalanine peptides three template structures were revealed. All the template structures have distinct peptide-turn features. The conformations in these N-aryl peptides were compared to those in the commonly studied N-methyl peptide analogues. We observed that the N-aryl peptides exhibit a considerable conformational homogeneity, and their conformations differ significantly from those in N-methyl analogues. We anticipate that the N-arylation of backbone amides has the potential for application as a novel tool for conformation and physicochemical modification in peptides.

New Hypothesis Insight of Structural Properties on Four Tau Peptide Fragments in Alzheimer's Disease: Origin from the Histidine Behaviors.

During protein folding and misfolding, structural properties and aggregation tendency can be significantly influenced by histidine behaviors (tautomeric behaviors and protonation behaviors). The original reasons were derived from the net charge changes and the various N/N-H orientation on imidazole rings. In the current study, total 18 independent REMD simulations were performed to investigate the histidine behaviors on four Tau peptide fragments (MBD, including R1, R2, R3, and R4 fragments). We found that, compared to R1, R2, R3 except (εδ), and R4 systems with flexible structural features, only R3(εδ) has dominating conformational structure (possibility of 81.3%) with three β-strand structures in parallel β-sheet structures at I4-K6and I24-H26, as well as antiparallel β-sheet structure at G19-L21. Importantly, the H25 and H26 residues (in R3(εδ) system) are directly involved in the sheet structure formations and strong H-bonded interactions (possibility range of 31.3%-44.7%). Furthermore, the donors and acceptors analysis confirmed that only R3(εδ) shows faraway amino acids interaction features in both H25 and H26 residues, and such cooperation effects of two histidine residues contribute to current structural features. The current study will be helpful to further enrichment of the histidine behavior hypothesis, it provides new insight for understanding protein folding and misfolding.

A New Class of Graph Grammars and Modelling of Certain Biological Structures

Graph grammars can be used to model the development of diverse graph families. Since their creation in the late 1960s, graph grammars have found usage in a variety of fields, such as the design of sophisticated computer systems and electronic circuits, as well as visual languages, computer animation, and even the modelling of intricate molecular structures Replacement of edges and nodes are the two primary approaches of graph rewriting. In this paper we introduce a new type of node replacement graph grammar known as nc-eNCE graph grammar. With this new class of graph grammars we generated certain graph classes and we showed that these class of graph grammars are more powerful than the existing edge and node controlled embedding graph grammars. In addition, these graph grammars were used to model several common protein secondary structures such as parallel and anti-parallel β-sheet structures in different configurations. The use of these graph grammars in modelling other bio-chemical structures and their interactions remains to be explored.

The effect of histidine behaviors on the structural properties of Aβ(1-42) peptide in protonation stage one, two, and three.

Histidine behaviors (including tautomeric and protonation behaviors, and integration on ε, δ, or p states) have been linked to protein folding and misfolding. However, the histidine behaviors of Aβ(1-42) are unconfirmed, which is the key point to understanding the pathogenesis of Alzheimer's disease. In the current study, 19 replica exchange molecular dynamics (REMD) simulations were performed to investigate the impact of histidine on the structural properties in protonation evolution stages one, two, and three. In contrast to the deprotonated (εεε) state, our current findings demonstrate that any protonated state will promote the formation of the β-sheet structure. The sheet-rich structures of (pεε), (δεp), (pεp), and (ppp) have the same basic characteristics of three-strand structures between the N-terminus, central hydrophobic core (CHC), and C-terminus. We found that the (pεε) (probability of 77.7%) and (δεp) (probability of 60.2%) prefer the abundant conformation over the other systems with higher regularity antiparallel β-sheet structure characteristics. Further H-bonding results indicate that H6 and H14 are more essential than H13. In addition, the Pearson correlation coefficient analysis revealed that the experimental result coincided with our simulated (δpε) system. The current study aids in the better understanding of the mechanisms of histidine behaviors, providing a fresh outlook on protein folding and misfolding.

Self-assembly of wide peptide nanoribbons via the formation of nonpolar zippers between β-sheets

Because zipper motifs between complementary sidechains can allow molecular order to be maintained over larger length scales, they hold great promise for the preparation of hierarchical peptide self-assemblies with long-range ordering. Although polar zippers based on sidechain-sidechain hydrogen bonding have been demonstrated to promote significant lamination of β-sheets into wide and flat nanoribbons, it remains a challenge to achieve such well-defined nanostructures through nonpolar zippers. In this study, we designed a series of short amphiphilic peptides Ac-I3XGK-NH2, where X at the intramolecular hydrophobic/hydrophilic interface denotes different hydrophobic residues. Microscopic imaging and neutron scattering measurements indicated that only Ac-I3VGK-NH2 could self-assemble into wide and uniform nanoribbons. Spectroscopic and solid-state NMR analyses revealed that the peptide adopted an anti-parallel β-sheet secondary structure with a two-residue shifting. Under such a molecular conformation and alignment, homogenous and extensive nonpolar zippers between β-sheets, consisting of alternating Val4-Ile3 and Ile2-Ile1 sidechain contacts, would be formed, leading to significant β-sheet lamination that can cause wide and flat nanoribbon assembly. Finally, the failure of the peptide variants to form wide and flat nanoribbons indicated a stringent requirement of extensive nonpolar zipper formation between β-sheets for primary sequence, especially the interaction mode of hydrophobic sidechains.

Effect of wheat bran dietary fiber on structural properties and hydrolysis behavior of gluten after synergistic fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae.

The effect of synergistic fermentation of Lactobacillus plantarum and Saccharomyces cerevisiae on the structural properties and aggregation behavior of gluten containing different wheat bran dietary fiber (WBDF) levels (0, 3, 6, 9, and 12%) was investigated. The results showed that WBDF addition affected the aggregation behavior of gluten at the molecular level, while WBDF significantly induced depolymerization behaviors in large aggregated gluten proteins (Molecular weight > 130 kDa) under reducing conditions (p < 0.05). In terms of secondary structure, WBDF significantly reduced glutamine side chain levels and reduced antiparallel β-sheet structures from 28.57 to 24.53% (p < 0.05). In addition, WBDF thermal properties and its water holding capacity were the main factors causing changes in thermal properties in the overall gluten system. This study provides new data for the improved production of sourdough whole grain and/or high fiber flour products.

Synthesis and Characterization of Propeller- and Parallel-Type Full-Length Amyloid β40 Trimer Models.

Oligomers of the amyloid β (Aβ) protein play a critical role in the pathogenesis of Alzheimer's disease. However, their heterogeneity and lability deter the identification of their tertiary structures and mechanisms of action. Aβ trimers and Aβ dimers may represent the smallest aggregation unit with cytotoxicity. Although propeller-type trimer models of E22P-Aβ40 tethered by an aromatic linker have recently been synthesized, they unexpectedly exhibited little cytotoxicity. To increase the flexibility of trimeric propeller-type models, we designed and synthesized trimer models with an alkyl linker, tert-butyltris-l-alanine (tButA), at position 36 or 38. In addition, we synthesized two parallel-type trimer models tethered at position 38 using alkyl linkers of different lengths, α,α-di-l-norvalyl-l-glycine (di-nV-Gly) and α,α-di-l-homonorleucyl-l-glycine (di-hnL-Gly), based on the previously reported toxic dimer model. The propeller-type E22P,V36tButA-Aβ40 trimer (4), which was designed to mimic the C-terminal anti-parallel β-sheet structures proposed by the structural analysis of 150 kDa oligomers of Aβ42, and the parallel-type E22P,G38di-nV-Gly-Aβ40 trimer (6) showed significant cytotoxicity against SH-SY5Y cells and aggregative ability to form protofibrillar species. In contrast, the E22P,G38tButA-Aβ40 trimer (5) and E22P,G38di-hnL-Gly-Aβ40 trimer (7) exhibited weak cytotoxicity, though they formed quasi-stable oligomers observed by ion mobility-mass spectrometry and native polyacrylamide gel electrophoresis. These results suggest that 4 and 6 could have some phase of the structure of toxic Aβ oligomers with a C-terminal hydrophobic core and that the conformation and/or aggregation process rather than the formation of stable oligomers contribute to the induction of cytotoxicity.

Identification of a Novel Antimicrobial Peptide From the Ancient Marine Arthropod Chinese Horseshoe Crab, Tachypleus tridentatus.

Humoral immunity is the first line of defense in the invertebrate immune system, and antimicrobial peptides play an important role in this biological process. A novel antimicrobial peptide, termed Tatritin, was identified and characterized in hemolymph of Chinese horseshoe crab, Tachypleus tridentatus, infected with Gram-negative bacteria via transcriptome analysis. Tatritin was significantly induced by bacterial infection in hemolymph and gill. The preprotein of Tatritin consists of a signal peptide (21 aa) and a mature peptide (47 aa) enriched by cysteine. The putative mature peptide was 5.6 kDa with a theoretical isoelectric point (pI) of 9.99 and showed a α-helix structure in the N-terminal and an anti-parallel β-sheet structure in the cysteine-stabilized C-terminal region. The chemically synthesized peptide of Tatritin exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Furthermore, Tatritin may recognize and inhibit pathogenic microorganisms by directly binding to LPS, DNA, and chitin. In addition, administration of Tatritin reduced the mortality of zebrafish after bacterial infection. Due to its broad-spectrum antimicrobial activity in vivo and in vitro and the sensitivity to drug-resistant bacterial strains, Tatritin peptide can be used as a new type of drug for infection treatment or as an immune enhancer in animals.

Topochemical Synthesis of a Heterochiral Peptide Polymer in Different Polymorphic Forms from Crystals and Aerogels.

We have designed a heterochiral dipeptide monomer (N3 -D-Ala-L-Val-NHCH2 C≡CH) modified with an azide group and an alkyne at its termini for topochemical azide-alkyne cycloaddition (TAAC) polymerization. We obtained two different polymorphs: PI (P21 ) from a DCM-toluene mixture, and PII (P-1) from a hexane-ethyl acetate mixture. PI adopts parallel β-sheet packing, and PII adopts antiparallel β-sheet packing. In both the cases, molecules from adjacent β-stacks are arranged in a head-to-tail manner. On heating, the polymorphs underwent TAAC reaction to form linear polymers having parallel β-sheet-like ordering (PI) and an antiparallel β-sheet structure (PII). PI reacted spontaneously at room temperature and the crystals showed cracking after the reaction, but PII was intact even after complete reaction. The dipeptide congealed various solvents, and the aerogels were identical to PI. These aerogels also underwent spontaneous TAAC reaction at room temperature to yield a pseudoprotein with alternate D- and L-amino acids.

S-Benzyl cysteine based cyclic dipeptide super hydrogelator: Enhancing efficacy of an anticancer drug via sustainable release.

Peptide-based low molecular weight supramolecular hydrogels hold promising aspects in various fields of application especially in biomaterial and biomedical sciences such as drug delivery, wound healing, tissue engineering, cell proliferation, and so on due to their extreme biocompatibility. Unlike linear peptides, cyclic peptides have more structural rigidity and tolerance to enzymatic degradation and high environmental stability which make them even better candidates for the above-said applications. Herein, a new small cyclic dipeptide (CDP) cyclo-(Leu-S-Bzl-Cys) (P1) consisting of L-leucine and S-benzyl protected L-cysteine was reported which formed a hydrogel at physiological conditions (at 37°C and pH = 7.46). The hydrogel formed from the cyclic dipeptide P1 showed very good tolerance towards environmental parameters such as pH and temperature and was seen to be stable for more than a year without any deformation. The hydrogel was thermoreversible and stable in the pH range 6-12. Mechanical strength of P1 hydrogel was measured by rheology experiments. Atomic force microscopy (AFM) and field emission scanning electron microscopy (FE-SEM) images revealed that, in aqueous solvents, P1 self-assembled into a highly cross-linked nanofibrillar network which immobilized water molecules inside the cages and formed the hydrogel. The self-assembled cyclic dipeptide acquired the antiparallel β-sheet secondary structure, which was evident from CD and Fourier transform infrared (FT-IR) studies. The β-sheet arrangement and formation of amyloid fibrils were further established by ThT binding assay. Furthermore, P1 was able to form a hydrogel in the presence of the anticancer drug 5-fluorouracil (5FU), and sustainable release of the drug from the hydrogel was measured in vitro. The hydrogelator P1 showed almost no cytotoxicity towards the human colorectal cancer cell line HCT116 up to a considerably high concentration and showed potential application in sustainable drug delivery. The co-assembly of 5FU and P1 hydrogel exhibited much better anticancer activity towards the HCT116 cancer cell line than 5FU alone and decreased the IC50 dose of 5FU to a much lower value.

Front Cover: Helical Chirality of Ferrocene Moieties in Cyclic Ferrocene‐Peptide Conjugates (Eur. J. Inorg. Chem. 5/2022)

The Front Cover shows how the helical chirality of ferrocene moieties in a series of ferrocene-peptide conjugates can be controlled by adjusting the conformational flexibility of the peptide chains through cyclization regulations. Cyclization regulations, which act as a clamp to tie the terminals of the peptide chains together, is one simple strategy to restrict the conformational flexibility of the peptide chains, resulting in the reordering of the hydrogen bonding interactions to switch the helical chirality of the ferrocene moiety. The helical chirality of the ferrocene moiety can be inverted without changing the chirality of the adjacent amino acids by changing the hydrogen bonding pattern exemplified by an antiparallel β-sheet type structure to a different secondary structure. More information can be found in the Review by T. Moriuchi.

Heterochirality-Mediated Cross-Strand Nested Hydrophobic Interaction Effects Manifested in Surface-Bound Peptide Assembly Structures.

Amino acid chirality has been envisioned as an important strategy to regulate structure and function of peptide self-assembled architectures. However, the molecular mechanism of chirality effects in peptide assemblies remains largely elusive. Here, the assembly structures of l-peptide polyphenylalanine F10 (FFFFFFFFFF) and block heterochiral peptide F5f5 (FFFFFfffff) composed of two FFFFF repeat blocks with opposite chirality were characterized at the single-molecule level by using scanning tunneling microscopy. Each peptide formed two distinctively different assembly structures on the HOPG surface, in which peptide chains took parallel and antiparallel β-sheet conformations, respectively. The molecular-level observations revealed that the staggered arrangement of cross-strand side chains achieved in the antiparallel β-sheet structure of the block heterochiral peptide facilitated intimate packing of side chains and maximized inter-residue van der Waals interactions, which led to more residues participating in assembly and greatly stabilized the β-sheet structure of the surface-bound peptide assembly, but such cross-strand nested interactions were not accessible in the heterochiral parallel β-sheet structure and the enantiomerically pure assembly structures. This work could contribute to the molecular insights of stereochemical interactions in peptide assemblies and feasibility of extending this block heterochirality pattern to other peptides with various lengths and amino acid compositions for structural regulations.

FTIR spectroscopy confirms age-related changes in protein conformation in a new independent dataset of human plasma samples

The loss of proteostasis is a key hallmark of aging and has been well documented using several model systems. However, in biofluids such as blood plasma, little is known about how the loss of proteostasis affects protein conformation during aging. Our previous work has demonstrated age-related changes in protein conformation in human plasma samples using FTIR spectroscopy and conformation-specific antibodies. To prove the potential of spectroscopy to detect slight changes in protein secondary structure during aging, we have applied the same methodology to a new and independent set of 24 human plasma samples from donors with ages ranging from 40 to 75 years. The results clearly show that from middle to old age there is a decrease in antiparallel β-sheet structures and an increase in intermolecular β-sheets, indicative of an increase in aggregation-prone proteins in human plasma over time. This confirms the potential of FTIR spectroscopy as a technique to evaluate protein conformational changes related to health and disease and as a suitable method to be used in a clinical setting to produce quick and reliable results.