Cristiano Luis Dias

Here, we report the purification and characterization of a haloacid dehalogenase type II (HAD-II) enzyme capable of direct and cell-free enzymatic defluorination by cleaving the resilient C-F bond in perfluorooctanoic acid (PFOA). While conventional remediation strategies rely on energy-intensive chemical/thermal methods, biological alternatives are limited by long whole-microbiome incubations and poorly understood metabolic pathways. We discovered a novel HAD-II enzyme from Achromobacter mucicolens found in PFAS-contaminated lacustrine sediment, providing evidence of real-time microbial adaptation. Within 24-hour incubation, the system released approximately 0.55 ppm fluoride (17% yield) from a 5ppm PFOA (equivalent to maximum fluoride of 3.24 ppm) in recombinant enzyme assays. Structural and phylogenetic analyses reveal that the newly discovered HAD-II belongs to a deeply divergent lineage sharing only 25% sequence identity with the previously characterized Delftia homologue while preserving the core HAD-like catalytic fold. Comparative molecular docking further elucidated this functional divergence, demonstrating that PFOA adopts a productive binding orientation near the conserved catalytic Asp15 within the A. mucicolens active-site pocket, whereas the Delftia counterpart forces non-productive binding outside the catalytic site. Together, our work unveils a previously unrecognized Achromobacter -associated dehalogenase that mediates PFAS defluorination despite severe sequence divergence, offering a critical new paradigm for targeted biological remediation.

Capsaicin is a natural bioactive compound found in chili peppers, with promising potential in various pharmacological applications, including analgesic, antipruritic, anti-inflammatory, anticancer, and antioxidant effects. Its clinical use, however, remains limited due to poor bioavailability, low aqueous solubility, and limited stability. To address these challenges, loading capsaicin in a small liposome bilayer has been proposed to enhance its transport across biological membranes. In this work, we perform molecular dynamics (MD) simulations to study the behavior of capsaicin in lipid bilayers. Two lipid types, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC), are used to investigate the effects of unsaturation in the lipid tails. Simulations were conducted under two initial conditions: (1) capsaicin molecules placed in the aqueous phase to study permeation behavior and (2) capsaicin preinserted into the bilayer to assess its equilibrium positioning and to study its behavior in the membrane environment. Our results show that capsaicin tends to aggregate in water with only small aggregates capable of passive permeation into the membrane. The POPC bilayer exhibits a greater capsaicin uptake than the DPPC bilayer, which is attributed to its larger area per lipid. Mass-density profiles indicate the preferred localization of capsaicin within the bilayers, which is consistent with the minimum free energy observed in the potential of mean force (PMF) profiles for translocated molecules into the bilayer. Notably, capsaicin does not significantly change the bilayer thickness and area per lipid when the bilayer is in the liquid phase. It decreases the lipid area per lipid, while maintaining a stable area per capsaicin. The estimated area per capsaicin molecule within the bilayer is 0.415 ± 0.010 nm². Interestingly, when the bilayer was in the gel phase, the ordered lipid was disrupted by the capsaicin. These findings provide valuable insights into the membrane interactions of capsaicin and support the rational design of liposomal delivery systems to enhance its pharmaceutical potential.

Supramolecular β-sheet peptide nanomaterials are of critical interest due to their relevance in amyloid disorders and are increasingly valued for applications in regenerative medicine, tissue engineering, and antimicrobial design. Amphipathic peptides, particularly those with alternating hydrophobic and hydrophilic residues, readily form amyloid-like pleated β-sheet fibrils. It has been demonstrated that the amino acid sequence order of isomeric peptides dramatically influences the self-assembly propensity of the resulting sequences as well as the morphology of the assembled pleated β-sheet nanomaterials. This was substantiated by our previous investigations of the peptides Ac-(FKFE)₂-NH₂ (L1), Ac-(FK)₂(FE)₂-NH₂ (L2), Ac-KE(F)₄KE-NH2 (L3), Ac-(KFFE)₂-NH₂ (L4), and Ac-FF(KE)₂FF-NH₂ (L5). Recently, interest in the Pauling and Corey rippled β-sheet motif, composed of coassembled enantiomeric l- and d-peptides in which the l- and d-enantiomers are organized in an alternating fashion, has been revitalized, although understanding of the rippled β-sheet fold lags far behind that of the naturally occurring pleated β-sheet. Herein, we interrogate the scope of rippled β-sheet formation by extending our previous study of the L1–L5 peptides to enantiomeric mixtures of these sequences to understand the effect of sequence order on rippled β-sheet formation. These integrated experimental and computational studies confirm that enantiomeric mixtures of these peptides have a significantly higher propensity to coassemble into putative rippled β-sheets than single enantiomers have to self-assemble into pleated β-sheets under the same solvent and concentration conditions. These findings extend our understanding of the rippled β-sheet motif and highlight the potential to exploit stereochemically diverse peptides in the design of next-generation biomaterials.

The presence of oxygen molecules (O₂) in biological membranes promotes lipid peroxidation of phospholipids with unsaturated acyl chains. On the other hand, cholesterol is considered to be an antioxidant molecule as it has a significant barrier effect on the permeation of O₂ across membranes. However, a comprehensive explanation of how cholesterol affects the distribution and diffusion of O₂ within lipid bilayers is yet to be established. In this study, we investigated the interaction of oxygen molecules with polyunsaturated lipid bilayers using molecular dynamics (MD) simulations. The degree of lipid unsaturation and the concentration of cholesterol were varied to study the permeation of O₂. The free energy profile of O₂ diffusing from the water phase to the lipid bilayer was calculated using biased umbrella MD simulations. The results show that O₂ passively translocates into the membrane without changing the physical properties of the bilayer. Interestingly, in the unsaturated lipid bilayers the presence of cholesterol led to a significantly decreased permeation of O₂ and an increase in the lipid chain order. Our results indicate that the hydroxyl groups of cholesterol strongly interact with the O₂ molecules effectively inhibiting interactions between the oxygens and the double bonds in unsaturated lipid tails. In addition, a linear relationship between permeation and the ratio of membrane thickness and area per lipid was found. These insights can help our understanding of how the degree of unsaturation in a lipid tail and cholesterol affect lipid peroxidation at the molecular level.

One of the mechanisms accounting for the toxicity of amyloid peptides in diseases like Alzheimer's and Parkinson's is the formation of pores on the plasma membrane of neurons. Here, we perform unbiased all-atom simulations of the full membrane damaging pathway, which includes adsorption, aggregation, and perforation of the lipid bilayer accounting for pore-like structures. Simulations are performed using four peptides made with the same amino acids. Differences in the nonpolar-polar sequence pattern of these peptides prompt them to adsorb into the membrane with the extended conformations oriented either parallel [peptide labeled F1, Ac-(FKFE)₂-NH₂], perpendicular (F4, Ac-FFFFKKEE-NH₂), or with an intermediate orientation (F2, Ac-FFKKFFEE-NH₂, and F3, Ac-FFFKFEKE-NH₂) in regard to the membrane surface. At the water-lipid interface, only F1 fully self-assembles into β-sheets, and F2 peptides partially fold into an α-helical structure. The β-sheets of F1 emerge as electrostatic interactions attract neighboring peptides to intermediate distances where nonpolar side chains can interact within the dry core of the bilayer. This complex interplay between electrostatic and nonpolar interactions is not observed for the other peptides. Although β-sheets of F1 peptides are mostly parallel to the membrane, some of their edges penetrate deep inside the bilayer, dragging water molecules with them. This precedes pore formation, which starts with the flow of two water layers through the membrane that expand into a stable cylindrical pore delimited by polar faces of β-sheets spanning both leaflets of the bilayer.

Protein self-assembly plays an important role in biological systems, accounting for the formation of mesoscopic structures that can be highly symmetric as in the capsid of viruses or disordered as in molecular condensates or exhibit a one-dimensional fibrillar morphology as in amyloid fibrils. Deposits of the latter in tissues of individuals with degenerative diseases like Alzheimer's and Parkinson's has motivated extensive efforts to understand the sequence of molecular events accounting for their formation. These studies aim to identify on-pathway intermediates that may be the targets for therapeutic intervention. This detailed knowledge of fibril formation remains obscure, in part due to challenges with experimental analyses of these processes. However, important progress is being achieved for short amyloid peptides due to advances in our ability to perform completely unbiased all-atom simulations of the self-assembly process. This perspective discusses recent developments, their implications, and the hurdles that still need to be overcome to further advance the field.

Amyloid fibrils have long been associated with many neurodegenerative diseases. The conventional picture of the formation and proliferation of fibrils from unfolded proteins comprises primary and secondary nucleation of oligomers followed by elongation and fragmentation thereof. In this work, we first employ extensive all-atom molecular dynamics (MD) simulations of short peptides to investigate the governing processes of fibril growth at the molecular scale. We observe that the peptides in the bulk solution can bind onto and subsequently diffuse along the fibril surface, which leads to fibril elongation via either bulk- or surface-mediated docking mechanisms. Then, to guide the quantitative interpretation of these observations and to provide a more comprehensive picture of the growth kinetics of single fibrils, a continuum model which incorporates the key processes observed in the MD simulations is formulated. The model is employed to investigate how relevant physical parameters affect the kinetics of fibril growth and identify distinct growth regimes. In particular, it is shown that fibrils which strongly bind peptides may undergo a transient exponential growth phase in which the entire fibril surface effectively acts as a sink for peptides. We also demonstrate how the relevant model parameters can be estimated from the MD trajectories. Our results provide compelling evidence that the overall fibril growth rates are determined by both bulk and surface peptide fluxes, thereby contributing to a more fundamental understanding of the growth kinetics of amyloid-like fibrils.

The formation of amyloid fibrils is a complex phenomenon that remains poorly understood at the atomic scale. Herein, we perform extended unbiased all-atom simulations in explicit solvent of a short amphipathic peptide to shed light on the three mechanisms accounting for fibril formation, namely, nucleation via primary and secondary mechanisms, and fibril growth. We find that primary nucleation takes place via the formation of an intermediate state made of two laminated β-sheets oriented perpendicular to each other. The amyloid fibril spine subsequently emerges from the rotation of these β-sheets to account for peptides that are parallel to each other and perpendicular to the main axis of the fibril. Growth of this spine, in turn, takes place via a dock-and-lock mechanism. We find that peptides dock onto the fibril tip either from bulk solution or after diffusing on the fibril surface. The latter docking pathway contributes significantly to populate the fibril tip with peptides. We also find that side chain interactions drive the motion of peptides in the lock phase during growth, enabling them to adopt the structure imposed by the fibril tip with atomic fidelity. Conversely, the docked peptide becomes trapped in a local free energy minimum when docked-conformations are sampled randomly. Our simulations also highlight the role played by nonpolar fibril surface patches in catalyzing and orienting the formation of small cross-β structures. More broadly, our simulations provide important new insights into the pathways and interactions accounting for primary and secondary nucleation as well as the growth of amyloid fibrils.

An understanding of how the amino acid sequence affects the interaction of peptides with lipid membranes remains mostly unknown. This type of knowledge is required to rationalize membrane-induced toxicity of amyloid peptides and to design peptides that can interact with lipid bilayers. Here, we perform a systematic study of how variations in the sequence of the amphipathic Ac-(FKFE)₂-NH₂ peptide affect its interaction with zwitterionic lipid bilayers using extensive all-atom molecular dynamics simulations in explicit solvent. Our results show that peptides with a net positive charge bind more frequently to the lipid bilayer than neutral or negatively charged sequences. Moreover, neutral amphipathic peptides made with the same numbers of phenylalanine (F), lysine (K), and glutamic (E) amino acids at different positions in the sequence differ significantly in their frequency of binding to the membrane. We find that peptides bind with a higher frequency to the membrane if their positive lysine side chains are more exposed to the solvent, which occurs if they are located at the extremity (as opposed to the middle) of the sequence. Non-polar residues play an important role in accounting for the adsorption of peptides onto the membrane. In particular, peptides made with less hydrophobic non-polar residues (e.g., valine and alanine) are significantly less adsorbed to the membrane compared to peptides made with phenylalanine. We also find that sequences where phenylalanine residues are located at the extremities of the peptide have a higher tendency to be adsorbed.