Substrate Interactions Research Articles

Distal protein-protein interactions contribute to nirmatrelvir resistance

SARS-CoV-2 main protease, Mpro, is responsible for processing the viral polyproteins into individual proteins, including the protease itself. Mpro is a key target of anti-COVID-19 therapeutics such as nirmatrelvir (the active component of Paxlovid). Resistance mutants identified clinically and in viral passage assays contain a combination of active site mutations (e.g., E166V, E166A, L167F), which reduce inhibitor binding and enzymatic activity, and non-active site mutations (e.g., P252L, T21I, L50F), which restore the fitness of viral replication. To probe the role of the non-active site mutations in fitness rescue, here we use an Mpro triple mutant (L50F/E166A/L167F) that confers nirmatrelvir drug resistance with a viral fitness level similar to the wild-type. By comparing peptide and full-length Mpro protein as substrates, we demonstrate that the binding of Mpro substrate involves more than residues in the active site. Particularly, L50F and other non-active site mutations can enhance the Mpro dimer-dimer interactions and help place the nsp5-6 substrate at the enzyme catalytic center. The structural and enzymatic activity data of Mpro L50F, L50F/E166A/L167F, and others underscore the importance of considering the whole substrate protein in studying Mpro and substrate interactions, and offers important insights into Mpro function, resistance development, and inhibitor design.

Borophene bottom-up syntheses: a critical review

Abstract The ever-expanding portfolio of two-dimensional (2D) materials now also includes metallic boron sheets, known as borophene. Initially predicted by simulation in 1997, borophene was first synthesized in 2015. This 2D form of boron exists under various polymorphic structures, the most observed being β_12 and χ_3. Borophene fills a crucial gap in the 2D material repertoire thanks to its intrinsic metallicity and relative stability in air. However, in view of practical applications including borophene, it is crucial to master its synthesis to enable controlling its structure and thus tuning its properties. This review focuses on its current bottom-up syntheses, i.e., physical vapor deposition and chemical vapor deposition. Indeed, borophene’s structure is different from the bulk boron one, which excludes its top-down synthesis. The impact of substrate characteristics, temperature, pressure conditions, and precursor molecules on borophene's phase and domain size is analyzed, along with key characterization techniques. The review extends to hydrogenated borophene and bilayer borophene for a comprehensive understanding of boron bonding and substrate interactions. Given the importance of ambient stability and transferability of 2D boron layers for device applications, a thorough comprehension of borophene oxidation is imperative. The review thus also discusses potential solutions to mitigate oxidation-related challenges.

Structural analysis of extracellular ATP-independent chaperones of streptococcal species and protein substrate interactions.

Streptococcal species are a leading cause of lower respiratory infections that annually affect millions of people worldwide. During infection, streptococcal species secrete a medley of virulence factors that allow the bacteria to colonize and translocate to deeper tissues. In many gram-positive bacteria, virulence factors are secreted from the cytosol across the bacterial membrane in an unfolded state. The bacterial membrane-cell wall interface is exposed to the potentially harsh extracellular environment, making it difficult for native virulence factors to fold before being released into the host. ATP-independent PPIase-type chaperones, PrsA and SlrA, are thought to facilitate folding and stabilization of several unfolded proteins to promote the colonization and spread of streptococci. Here, we present crystal structures of the molecular chaperones of PrsA and SlrA homologs from streptococcal species. We provide evidence that the Streptococcus pyogenes SlrA homolog, PpiA, has PPIase activity and binds to cyclosporine A. In addition, we show that Streptococcus pneumoniae PrsA and SlrA directly interact and fold the cholesterol-dependent pore-forming toxin and critical virulence determinant, pneumolysin.

Implications of frequent hitter E3 ligases in targeted protein degradation screens.

Targeted protein degradation (TPD) offers a promising approach for chemical probe and drug discovery that uses small molecules or biologics to direct proteins to the cellular machinery for destruction. Among the >600 human E3 ligases, CRBN and VHL have served as workhorses for ubiquitin-proteasome system-dependent TPD. Identification of additional E3 ligases capable of supporting TPD would unlock the full potential of this mechanism for both research and pharmaceutical applications. This perspective discusses recent strategies to expand the scope of TPD and the surprising convergence of these diverse screening efforts on a handful of E3 ligases, specifically DCAF16, DCAF11 and FBXO22. We speculate that a combination of properties, including superficial ligandability, potential for promiscuous substrate interactions and high occupancy in Cullin-RING complexes, may position these E3 ligases as 'low-hanging fruit' in TPD screens. We also discuss complementary approaches that might further expand the E3 ligase landscape supporting TPD.

Structural Insights into Broad-Range Polyphosphate Kinase2‑II Enzymes Applicable for Pyrimidine Nucleoside Diphosphate Synthesis.

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP‑consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85% of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49%. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

Exploring the Potential of High-Power Impulse Magnetron Sputtering for Nitride Coatings: Advances in Properties and Applications

High-Power Impulse Magnetron Sputtering (HiPIMS) has emerged as an excellent technology for producing high-quality nitride coatings, such as aluminum nitride (AlN), titanium nitride (TiN), chromium nitride (CrN), and silicon nitride (SiN), and composite nitride coatings such as titanium aluminum nitride (TiAlN), TiAlNiCN, etc. These coatings are known for their exceptional hardness, thermal stability, and corrosion resistance. These make them ideal for high-performance applications. HiPIMS distinguishes itself by generating highly ionized plasmas that facilitate intense ion bombardment, leading to nitride films with superior mechanical strength, durability, and enhanced thermal properties compared to traditional deposition techniques. Critical HiPIMS parameters, including pulse duration, substrate bias, and ion energy, are analyzed for their influence on enhancing coating density, adhesion, and hardness. The review contrasts HiPIMS with other deposition methods, highlighting its unique ability to create dense, uniform coatings with improved microstructures. While HiPIMS offers substantial benefits, it also poses challenges in scalability and process control. This review addresses these challenges and discusses hybrid, bipolar, and synchronized HiPIMS solutions designed to optimize nitride coating processes. Hybrid HiPIMS, for instance, combines HiPIMS with other sputtering techniques like DCMS or RF sputtering to achieve balanced deposition rates and high-quality film properties. Bipolar HiPIMS enhances process stability and film uniformity by alternating the polarity, which helps mitigate charge accumulation issues. Synchronized HiPIMS controls precise pulse timing to maximize ion energy impact and improve substrate interaction, further enhancing the structural properties of the coatings. Hence, to pave the way for future research and development in this area, insights of the HiPIMS have been presented that underline the role of HiPIMS in meeting the demanding requirements of advanced industrial applications. Overall, this review article comprehensively analyzes the recent strategies and technological innovations in HiPIMS and highlights the significant potential of HiPIMS for advancing the nitride coating field.

Lattice-guided growth of dense arrays of aligned transition metal dichalcogenide nanoribbons with high catalytic reactivity.

Transition metal dichalcogenides (TMDs) exhibit unique properties and potential applications when reduced to one-dimensional (1D) nanoribbons (NRs), owing to quantum confinement and high edge densities. However, effective growth methods for self-aligned TMD NRs are still lacking. We demonstrate a versatile approach for lattice-guided growth of dense, aligned MoS2 NR arrays via chemical vapor deposition (CVD) on anisotropic sapphire substrates, without tailored surface steps. This method enables the synthesis of NRs with widths below 10 nanometers and longitudinal axis parallel to the zigzag direction, being also extensible to the growth of WS2 NRs and MoS2-WS2 heteronanoribbons. Growth is influenced by both substrate and CVD temperature, indicating the role of anisotropic precursor diffusion and substrate interaction. The 1D nature of the NRs was asserted by the observation of Coulomb blockade at low temperatures. Pronounced catalytic activity was observed at the edges of the NRs, indicating their promise for efficient catalysis.

Stretchable, Self-Healable, and Durable Conductive Elastomer Derived from a Rationally Designed Covalently Cross-Linked Network.

Silver nanowire (Ag NW)-based elastic conductors have been considered a promising candidate for key stretchable electrodes in wearable devices. However, the weak interface interaction of Ag NWs and elastic substrates leads to poor durability of electronic devices. For everyday usage, an additional self-healing ability is required to resist scratching and damage. Therefore, robust Ag NW-based elastic conductors possessing stretchability, self-healing, and stability are highly desirable and challenging. Here, we present a universal interface tailoring strategy that introduces thiols onto the dynamically cross-linked elastic substrate surface. The surface thiol groups strongly interact with Ag NWs through Ag-S bonds, forming the stable conductive layer on the elastic substrate. At elevated temperatures, the Ag NWs are partially embedded in the surface of the elastic substrate and a buffer layer is formed to release the concentrated stress. As a result, the formed Ag NW-based elastic conductor displays the combination of good conductivity, high stretchability (>1000%), efficient self-healing capability (>95%), and remarkable stability. Besides, the Ag NW-based elastic conductor combining the good properties mentioned above is suitable for fabricating a sensitive and durable strain sensor against cyclic strain. The presented strategy can be considered a versatile and effective route to generating other surface thiol-rich covalently cross-linked elastomers with dynamic disulfide bonds.

Efficient Cytosolic Delivery of Single-Chain Polymeric Artificial Enzymes for Intracellular Catalysis and Chemo-Dynamic Therapy.

Designing artificial enzymes for in vivo catalysis presents a great challenge due to biomacromolecule contamination, poor biodistribution, and insufficient substrate interaction. Herein, we developed single-chain polymeric nanoparticles with Cu/N-heterocyclic carbene active sites (SCNP-Cu) to function as peroxidase mimics for in vivo catalysis and chemo-dynamic therapy (CDT). Compared with the enzyme mimics based on unfolded linear polymer scaffold and multichain cross-linked scaffold, SCNP-Cu exhibits improved tumor accumulation and CDT efficiency both in vitro and in vivo. Protein-like size of the SCNP scaffold promotes passive diffusion, whereas positive surface charge allows its active transcytosis for deep tumor penetration and hence accumulation in the tumor site. The submolecular compartments of the SCNP scaffold effectively protect the active sites from protein bindings, thereby providing a "cleaner" microenvironment for catalysis within a living system. The folded structure of SCNP-Cu facilitates their cytosolic delivery of and free diffusion within cytosol, ensuring efficient contact with endogenous H2O2, in situ generation of toxic hydroxyl radicals (·OH), and effective damage of intracellular targets (i.e., lipids, nucleic acids). This work establishes versatile SCNP-based nanoplatforms for developing artificial enzymes for in vivo catalysis.

Protein S-palmitoylation regulates the virulence of plant pathogenic fungi.

Protein S-palmitoylation, a universal posttranslational modification catalyzed by a specific group of palmitoyltransferases, plays crucial roles in diverse biological processes across organisms by modulating protein functions. However, its roles in the virulence of plant pathogenic fungi remain underexplored. In a recent study, Y. Duan, P. Li, D. Zhang, L. Wang, et al. (mBio 15:e02704-24, 2024, https://doi.org/10.1128/mbio.02704-24) reported that the palmitoyltransferases UvPfa3 and UvPfa4 regulate the virulence of the rice false smut pathogen Ustilaginoidea virens. Through comprehensive characterization of S-palmitoylation sites, they revealed that S-palmitoylated proteins in U. virens are enriched in mitogen-activated protein (MAP) kinase and autophagy pathways, with MAP kinase UvSlt2 being a key target of UvPfa4-mediated S-palmitoylation. Further investigation demonstrated that S-palmitoylation of UvSlt2 is critical for its kinase activity, substrate interaction ability, and virulence function in U. virens. These findings reveal UvPfa4-mediated S-palmitoylation as a vital regulatory mechanism in U. virens virulence, highlighting the importance of protein S-palmitoylation in the pathogenicity of plant pathogenic fungi.

Unveiling Tetrafluoromethane Decomposition over Alumina Catalysts.

Tetrafluoromethane (CF4), the simplest perfluorocompound known as a "forever chemical", presents substantial environmental challenges due to its health risks and contribution to the greenhouse effect. Designing efficient catalysts for CF4 decomposition remains difficult, compounded by limited understanding of the mechanisms. Here, we use constrained ab initio molecular dynamics (cAIMD) simulations and in situ experiments to elucidate the mechanism of alumina-catalyzed CF4 decomposition, highlighting the pivotal role of surface hydroxyl groups. The initial C-F bond breaking is the rate-determining step, with surface hydroxyl groups reducing the reaction free energy from 1.69 to 1.34 eV. These hydroxyl groups also facilitate the self-healing of oxygen vacancies generated during the decomposition. Contrary to the belief that CF4 decomposes directly into CO2, our cAIMD simulations, supported by synchrotron vacuum ultraviolet photoionization mass spectrometry data, reveal significant CF2O and CO byproducts. Experimental data in an anhydrous environment indicate that water primarily replenishes surface hydroxyl groups rather than directly participating in decomposition. We conclude that the relatively high efficiency of Al2O3 catalysts stems from three key properties: (1) the presence of active sites with a specific affinity for CF4 adsorption, ensuring efficient substrate interaction; (2) appropriate metal-oxygen bond strength, enabling the participation of lattice oxygen in the reaction; and (3) a high density of surface hydroxyl groups that facilitate the initial C-F bond cleavage and the self-healing of oxygen vacancies.

Interactions between 3Al2O3·2SiO2, CA2, CA6, and Molten Slags

Herein, the interaction between the oxide substrates (mullite (3Al2O3·2SiO2), calcium aluminates CaO⋅2Al2O3 (CA2) and CaO⋅6Al2O3 (CA6), and the slags with various basicities (CaO/(Al2O3 + SiO2) (1.0, 0.9, and 0.8)) and MgO (5 and 10 wt%) is investigated. The contact angle of the molten slag on oxide substrates is found to be strongly regulated by the several factors including the chemical composition of the slag, the newly formed reaction product between molten slag and substrates, and the porosity of the substrates. For the case of mullite substrate, the slags infiltrated the substrates, with various morphologies of alumina observed within the penetrated layer of the mullite. In contrast, the CA2 substrate showed less penetration compared to the other substrates. Nevertheless, the newly generated CaAl2O4 phase was detected within the pores of CA2 substrate. During the interaction of slag and CA6 substrate, the CaAl2O4 and melilite phases are identified. The presence of MgO in the slag leads to the generation of MgAl2O4 phases. The corrosion depth is found to be enhanced via basicity. Furthermore, the solidified slags with 10 wt% MgO addition are found to have noticeable porosities. The porous structure is elucidated by the decreased slag density attributed to the generation of MgAl2O4 phase.

Anti-aggregation Properties of the Mini-Peptides Derived from Alpha Crystallin Domain of the Small Heat Shock Protein, Tpv HSP 14.3.

The highly conserved alpha crystallin domain of the small heat shock proteins is essential for dimerization and also implicated in substrate interaction. In this study, we designed four novel mini-peptides from alpha crystallin domain of archaeal Small Heat Shock Protein Tpv HSP 14.3. Among the peptide designs, the mini-peptides 38SDLVLEAEMAGFDKKNIKVS57 and 40LVLEAEMAGFD50 overlapped to the sequences of β3-β4 region. The other two peptides 77YIDQRVDKVYKVVKLPVE94 and 107GILTVRMK114 correspond to β6-β7 region and β9, respectively. Functional activity of the peptides was evaluated by monitoring heat-induced aggregation of the model substrates alcohol dehydrogenase at 43°C and citrate synthase at 45°C. Our results showed that the (38-57) and the (77-94) fragments exhibited chaperone activity with both of the substrate proteins. The (40-50) fragment while exhibiting a noticeable protective effect (> 90%) when tested with citrate synthase showed an anti-chaperone property toward alcohol dehydrogenase. Unlike the (40-50) fragment, the (107-114) fragment did not show any chaperone activity with citrate synthase but exhibited the highest chaperone efficiency among four mini-peptides with alcohol dehydrogenase. The selectivity of the (40-50) and the (107-114) fragments in targeting the client proteins is most likely dependent on their surface hydrophobicity and/or charge as revealed by the sequence and exposed surface analyses.

Membrane wetting by biomolecular condensates is facilitated by mobile tethers.

Biomolecular condensates frequently rely on membrane interactions for localization, recruitment, and chemical substrates. These interactions are often mediated by membrane-anchored tethers, a feature overlooked by traditional wetting models. Using a surface free-energy framework that couples surface tension with tether density, we solve for the contact angle and tether density in a spherical cap geometry, generalizing the Young-Dupré equation. While the contact angle retains its force-balance form, the tether density depends nontrivially on the form and strength of tether-condensate interactions. We solve for this dependence within a simple interaction model, and find a wetting phase diagram with a transition from non-wetting to partial to complete wetting over a biologically realistic parameter range. This work provides a quantitative framework for characterizing condensate-membrane interactions, uncovering potential mechanisms by which membranes mediate cellular organization and function.

From Chains to Arrays: Substrate-Mediated Self-Assembly of Diboron Molecules.

In this study, we explore the substrate-mediated control of self-assembly behavior in diboron molecules (C12H8B2O4, B2Cat2) using scanning tunneling microscopy (STM). The structural transformation of B2Cat2 molecules from one-dimensional (1D) molecular chains to two-dimensional (2D) molecular arrays was achieved by changing the substrate from Au(111) to bilayer graphene (BLG), highlighting the key role of substrate interactions in determining the assembly structure. Notably, the B-B bond in the molecular arrays on BLG is distinctly pronounced, reflecting a more refined molecular resolution with distinct electronic states than that on Au(111). Density functional theory (DFT) calculations confirm the weak interaction between B2Cat2 molecules and the BLG substrate, which facilitates the formation of 2D molecular arrays on BLG. This work demonstrates how controlling substrate properties enables the formation of 1D chains and 2D arrays, providing valuable insights for the design of next-generation molecular electronics and catalysis systems.

Characterization of a Dual Function Peptide Cyclase in Graspetide Biosynthesis.

Graspetides are a diverse family of ribosomally synthesized and post-translationally modified peptides with unique macrocyclic structures formed by ATP-grasp enzymes. Group 11 graspetides, including prunipeptin, feature both macrolactone and macrolactam cross-links. Despite the known involvement of a single ATP-grasp cyclase in the dual macrocyclizations of groups 5, 7, and 11 graspetides, detailed mechanistic insights into these enzymes remain limited. Here, we reconstructed prunipeptin biosynthesis from Streptomyces coelicolor using recombinant PruA and PruB macrocyclase. PruB exhibited kinetic behavior similar to other characterized graspetide cyclases, with a notably higher kcat, likely due to utilization of an ATP-regeneration system. The X-ray crystal structure of PruB revealed distinct features as compared to groups 1 and 2 enzymes. Site-directed mutagenesis identified critical roles of key residues for the PruB reaction, including the DxR motif conserved in other graspetide cyclases. Additionally, computational modeling of the PruA/PruB cocomplex uncovered substrate interactions and suggested that PruB first catalyzes a macrolactone bond formation on PruA. This study enhances our understanding of ATP-grasp enzyme mechanisms in graspetide biosynthesis and provides insights for engineering these enzymes for future applications.

3d oxide molecules to tailor large magnetic anisotropy energies on MgO films

Designing systems with large magnetic anisotropy energy (MAE) is desirable and critical for nanoscale magnetic devices. A recent breakthrough achieved the theoretical limit of the MAE for 3d transition metal atoms by placing a single Co atom on a MgO(100) surface, a result not replicated by standard first-principles simulations. Our paper, incorporating Hubbard-U correction and spin-orbit coupling, successfully reproduces and explains the high MAE of a Co adatom on a MgO (001) surface. We go further by exploring ways to enhance MAE in 3d transition metal adatoms through different structural geometries of 3d−O molecules on MgO. One promising structure, with molecules perpendicular to the surface, enhances MAE while reducing substrate interaction, minimizing spin fluctuations, and boosting magnetic stability. Additionally, we demonstrate significant control over MAE by precisely placing 3d−O molecules on the substrate at the atomic level. Published by the American Physical Society 2024

Two Isomeric Thienoacenes in Thin Films: Unveiling the Influence of Molecular Structure and Intermolecular Packing on Electronic Properties.

Isomerism of molecular structures is often encountered in the field of organic semiconductors, but little is known about how it can impact electronic and charge transport properties in thin films. This study reveals the molecular orientation, electronic structure, and intermolecular interactions of two isomeric thienoacenes (DN4T and isoDN4T) in thin films, in relation to their charge transport properties. Utilizing scanning tunneling microscopy (STM), angle-resolved photoemission spectroscopy (ARUPS), and near-edge X-ray absorption fine structure measurements (NEXAFS), we systematically analyze the behavior of these isomers from submonolayer to multilayer coverage on highly ordered pyrolytic graphite (HOPG) as substrates. We find that at submonolayer coverage both DN4T and isoDN4T molecules predominantly adopt a nearly flat-lying orientation on the surface, minimizing intermolecular interactions. The distinct emission features of the highest occupied molecular orbital (HOMO) level in ARUPS enables the determination of molecular reorganization energies. These are found to be in good agreement with theoretical predictions, suggesting superior charge transport in DN4T compared to isoDN4T. Notably, thickness-dependent photoemission measurements reveal a significant splitting (approximately 450 meV) of the HOMO level of isoDN4T, attributed to polarization-induced effects rather than wave function overlap, indicating a nuanced interplay between molecular packing and electronic properties. Our results underscore the importance of molecular packing and substrate interactions in determining the electronic structure and transport properties of organic semiconductor thin films. Substrate-induced polymorphism and the crucial role of polarization-induced effects influencing charge transport are highlighted. These insights are pivotal for future engineering of molecular and thin film structures, aiming to enhance the performance of organic semiconductor-based devices.

Modulating Biomimetic Peroxidase Activity of CuMOFs Through Ligand Engineering: Sensitive Colorimetric Detection of H2O2 and Glutathione

AbstractNatural peroxidase enzymes are widely utilized for various diagnostic applications, yet their inherent instability and susceptibility to external factors pose challenges. This study introduces a ligand engineering approach to fine‐tune the peroxidase enzyme‐like activity of copper‐based metal‐organic frameworks (MOFs). We have rationally modulated the enzyme‐mimicking activity of a series of Cu‐BDC‐X based MOFs via employment of various ligands (BDC = 1,4‐benzene dicarboxylic acid; CuMOF‐1, X═H2O; CuMOF‐2, X═4,4′‐bipyridine and CuMOF‐3, X═N‐methylimidazole). As compared to its analogues, CuMOF‐3 exhibited an enhanced catalytic activity, most likely due to N‐methylimidazole‐induced structural distortion that fine‐tunes the metal's electronic environment and improves substrate interactions. Additionally, this modification allowed CuMOF‐3 to be miniaturized to the nanoscale, enhancing its catalytic performance. Mechanistic investigations revealed the generation of •OH as reactive oxygen species (ROS) to accelerate tetramethylbenzidine oxidation, promoting peroxidase‐like activity. The observed catalytic behavior of CuMOF‐3 followed Michaelis–Menten kinetics, exhibiting lower Km value compared to those of natural peroxidase enzymes. Under optimized conditions, CuMOF‐3 facilitated the development of a colorimetric assay for sensitive detection of H2O2 and glutathione (GSH) in various spiked food samples. This study uniquely demonstrates the impact of tailored ligand combinations and MOF geometry on optimizing peroxidase‐mimicking activity, providing a new direction for the design of high‐performance colorimetric diagnostic tools in the food processing industry.

Mapping nanoparticle formation and substrate heating effects: a fluence-resolved approach to pulsed laser-induced dewetting

This study investigates the fluence-dependent evolution of gold nanoparticles formed through single nanosecond pulsed laser dewetting of a gold thin film on a fused silica substrate. By employing a well-defined Airy-like laser spatial profile and reconstructing scanning electron microscope images across the irradiation spot into a panoramic view, we achieve a detailed continuous analysis of the nanoparticle formation process. Our morphological analysis, combined with finite element thermal simulations directly correlated with the applied fluence, identifies two distinct thresholds. The first threshold corresponds to the dewetting of the gold film at its melting point, resulting in large, sparse nanoparticles. The second threshold, where the substrate temperature reaches values near its melting point, leads to the formation of numerous small nanoparticles and a significant increase in coverage area. Notably, the formation of these small nanoparticles is attributed to substrate heating, which alters the interaction between the molten gold film and the substrate, increasing adhesion. Contact angle measurements of the nanoparticles confirm this change, revealing a shift in wettability, and highlighting the crucial role of substrate heating in modulating the interactions leading to nanoparticle formation. Our findings underscore the intricate interplay between laser fluence, material properties, and substrate interactions in pulsed laser dewetting, with the well-defined laser profile offering valuable insights into these dynamics.