Study on the Interaction Mechanisms and Antibacterial Properties of BSA/LF with SAP and LF with DTAB

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To address the pressing need for highly effective, low-toxicity natural antimicrobials, this work systematically examines the multi-scale interactions and antibacterial properties of model protein bovine serum albumin (BSA) and functional protein lactoferrin (LF) with natural saponin (SAP), as well as LF with the cationic surfactant dodecyltrimethylammonium bromide (DTAB). By combining UV-Vis and fluorescence spectroscopy with molecular docking, we elucidate conformation-driven structural dynamics mediated by intermolecular forces. Hydrogen bonding and hydrophobic interactions cooperatively stabilize the BSA-SAP complex, leading to a reduction in α-helical content and increased hydrophobicity around tryptophan residues. LF–SAP association is governed by a combination of hydrogen bonding, van der Waals, and hydrophobic forces. Notably, DTAB binding to LF, driven by hydrogen bonding and hydrophobic interactions, induces significant conformational reorganization. Antibacterial assays demonstrate that the LF-DTAB complex exhibits strong synergistic activity against both E. coli and S. aureus , with notably enhanced efficacy against the latter. This selectivity is likely attributable to the DTAB-induced conformational state of LF and its differential affinity toward bacterial cell-wall components, possibly involving electrostatic and hydrophobic interactions. Our findings propose a protein-ligand synergy strategy to overcome limitations of single-agent antimicrobials, providing a molecular framework for the design of next-generation colloidal antimicrobial systems. Protein-bioactive interactions Antibacterial activity Molecular docking Conformational change Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Interactions between proteins and bioactive ligands represent a cornerstone of colloid science, with profound implications for understanding biological regulation and designing advanced functional materials [1, 2]. Amid the global challenge of antimicrobial resistance, this study leverages fundamental colloidal and interfacial principles to propose a synergistic protein-ligand strategy for next-generation antimicrobials. We systematically investigate multi-scale interactions and functional modulation in three model systems: the transport protein BSA with natural SAP, the multifunctional protein LF with SAP, and LF with the cationic surfactant DTAB. This comparative design allows us to uncover general principles of molecular assembly, conformational adaptation, and functional synergy that govern bio-colloidal systems, bridging the gap between natural and synthetic bioactive hybrids. The specific scientific questions we address are threefold and interconnected. First, the complexation of SAP, a potent but poorly soluble natural biosurfactant, with the model protein BSA represents a classic colloid delivery challenge. While BSA is an established carrier [3, 4], a mechanistic and quantitative understanding of the forces driving SAP-BSA association [5, 6], the resultant protein structural dynamics, and how this translates to modulated antimicrobial efficacy remains fragmented. Second, the interaction between SAP and the multifunctional [7, 8], inherently antimicrobial protein LF is virtually unexplored. The potential for synergy between the iron-sequestration mechanism of LF [9, 10] and the membrane-disruptive action of SAP is compelling [11, 12], yet the colloidal stability, binding landscape, and functional outcome of an LF-SAP composite are unknown. Third, the interaction between LF [13, 14] and the synthetic cationic surfactant DTAB sits at the core of colloid and interface science [15, 16]. Understanding how DTAB binding remodels LF conformation, perturbs its colloidal state, and reprograms its functionality is key to designing bio-surfactant hybrids that mitigate surfactant toxicity while amplifying and diversifying antimicrobial action [17]. To bridge these gaps, we employ an integrated structure-dynamics-function methodology. By combining UV-Vis and fluorescence spectroscopy with molecular docking, we decipher the hierarchy of intermolecular forces, quantify binding parameters, and map conformational transitions for each complex. Crucially, we directly correlate these molecular- and nano-scale insights with macroscopic functional performance through rigorous antibacterial assays. This work moves beyond descriptive interaction studies, establishing a colloid-science-guided framework for achieving programmable protein-ligand synergy. It provides a rational blueprint for designing next-generation antimicrobial materials, where controlled interfacial assembly and mechanistic cooperation are leveraged to overcome the limitations of single-agent systems. 2. Materials and methods 2.1 Materials BSA (analytical grade) and LF (analytical grade) were purchased from Macklin Inc. SAP (analytical grade) and DTAB (analytical grade) were obtained from Xiya Chemical Technology Co., Ltd. Phosphate buffered saline (PBS, pH 7.4, analytical grade) was sourced from Macklin Inc. The Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) were acquired from Qingdao Haibo Biotechnology Co., Ltd. Peptone, yeast extract powder, and agar powder (all analytical grade) were procured from Beijing AoBoXing Bio-Tech Co., Ltd. Other analytical-grade reagents were supplied by Beijing Innochem Science & Technology Co., Ltd. All reagents were used without further purification. Experimental systems and controls employed pH 7.4 PBS as solvent. Glassware was rinsed with triply distilled water (conductivity < 3 µS·cm⁻¹), and mixtures were prepared gravimetrically at predetermined ratios. 2.2 Measurement of fluorescence spectrum Fluorescence spectra were acquired using an FL-4600 spectrofluorometer equipped with a thermostatic controller and 10 mm quartz cuvette. Composite solution systems were prepared by mixing protein solutions with surfactant solutions at varying concentrations. Measurements were performed at 298.2 K with excitation fixed at 280 nm and emission scanned from 300 to 500 nm. 2.3 Measurement of UV-vis absorption spectrum UV-vis absorption spectrum of the composite solution was recorded using a UH4150 spectrophotometer equipped with a 10 mm quartz cuvette and Peltier temperature controller. Measurements were performed at 298.2 K with wavelength scanning from 200 to 350 nm. 2.4 Molecular docking Molecular docking was performed using the following protocol: The crystal structures of BSA (PDB ID: 4F5S) and LF (PDB ID: 3IB0) were retrieved from the Protein Data Bank (PDB) [18, 19, 20]. These receptor structures were prepared with AutoDock Tools through hydrogen addition, Gasteiger charge assignment, and PDBQT format conversion [21, 22]. Ligand structures of SAP and DTAB were obtained from PubChem, followed by protonation state optimization and energy minimization using Open Babel [23]. Docking grids were centered on predicted binding sites with dimensions 40×40×40 Å. Automated docking calculations were executed in AutoDock Vina and the optimal binding pose for each complex was selected based on the lowest binding energy [24]. Protein-ligand interactions were finally visualized and analyzed using PyMOL [25]. 2.5 Antibacterial testing S. aureus and E. Coli suspensions, each at a concentration of 10⁶ CFU/mL, were diluted 1000-fold in sterile phosphate-buffered saline (PBS) to obtain working suspensions [26]. Subsequently, 1 mL of each bacterial working suspension was spread evenly onto the surface of Tryptic Soy Agar (TSA) plates [27]. After allowing the inoculated plates to air-dry, sterile 6-mm-diameter filter paper discs were individually saturated with 1 µL of the test sample solution. The sample-saturated discs were then aseptically placed onto the inoculated TSA plates, ensuring the bacterial contact surface faced downward. Each plate was divided into six sectors: five sectors received discs saturated with test samples, while the remaining sector received a disc saturated solely with sterile PBS to serve as the negative control. All plates were incubated at 37°C for approximately 48 h. Following incubation, the zones of inhibition were observed, and their diameters were measured to assess antibacterial activity. The entire antibacterial assay procedure was conducted under aseptic conditions. 3. Results and Discussion 3.1 Analysis of fluorescence spectroscopy To investigate the interaction mechanisms within the composite systems, fluorescence spectroscopy was employed to monitor the influence of surfactants (SAP/DTAB) on the microenvironments surrounding amino acid residues in the proteins (BSA/LF) [28, 29]. Under conditions of fixed protein concentration, a systematic decrease in protein fluorescence emission intensity was observed across all systems (BSA-SAP, LF-SAP, LF-DTAB) with increasing concentrations of either SAP or DTAB in Fig. 1 . This indicates a significant fluorescence quenching effect induced by the surfactants on the proteins, confirming the presence of strong interactions between them. All protein samples exhibit a characteristic fluorescence emission peak near 280 nm corresponding to the typical emission peak of tryptophan residues [30]. Notably, distinct patterns in λ max shifts were observed among the different systems: In the BSA-SAP system, a slight blue shift in λ max occurred with increasing SAP concentration in Fig. 1 a. This shift suggests that the primary hydrophobic fluorophores in BSA, such as tryptophan (Trp) and tyrosine (Tyr) residues, gradually transitioned from the internal hydrophobic environment to a microenvironment of lower polarity. This directly reflects conformational rearrangements within the tertiary structure of BSA, demonstrating that SAP induces conformational changes in the protein. The stable λ max at 280 nm indicates that SAP binding does not perturb the hydrophobic microenvironment around tryptophan residues in LF in Fig. 1 b. This suggests that the SAP-LF interaction may be strong yet non-disruptive to core structure of LF, or that the binding site is distal to these residues. For the LF-DTAB system, a blue shift in λ max was also observed as the DTAB concentration increased in Fig. 1 c. This indicates that DTAB similarly induced alterations in the tertiary structure of LF, leading to increased exposure of its internal hydrophobic residues to a more hydrophobic microenvironment. Particularly noteworthy is that the decreasing trend in fluorescence intensity leveled off when the DTAB concentration reached 3.0×10⁻² mol/L, suggesting that the LF-DTAB complex likely reached a binding saturation state, forming a stable complex at this concentration. 3.2 Anaysis of ultraviolet absorption spectrum The UV absorption characteristics of proteins primarily arise from the π-π* electronic transitions in Trp, Tyr, and phenylalanine (Phe) residues, as well as peptide bonds [31]. Changes in UV absorption spectrum can sensitively reflect ligand-induced conformational alterations in proteins [32, 33]. As shown in Fig. 2 , both BSA and LF exhibited characteristic absorption peaks near ~ 280 nm in the absence of surfactants, attributed to the π-π* transitions of Tyr and Trp residues [34, 35]. With increasing concentrations of either SAP or DTAB, the absorbance of both proteins at 280 nm significantly increased. This indicates that the formation of surfactant-protein complexes led to the exposure of previously buried Trp/Tyr residues to the solvent environment, consequently enhancing the probability of π-π* transitions in their aromatic ring conjugated systems. This observation confirms conformational changes in the proteins. Further analysis revealed that the increasing SAP concentrations induced a slight blue shift in the maximum emission wavelength for both BSA-SAP and LF-SAP systems in both the BSA-SAP and LF-SAP systems, indicating that SAP modulates the microenvironment of aromatic amino acid residues in these proteins toward enhanced hydrophobicity and reduced polarity (Fig. 2 a and 2 b). In contrast, while the LF-DTAB system also showed increased UV absorption, its λ em remained unchanged (Fig. 2 c). Static quenching involves the formation of ground-state complexes and typically alters absorption spectra, whereas dynamic quenching generally does not affect absorption characteristics. Based on this cooperative response mechanism observed in UV and fluorescence spectra, it can be inferred that SAP quenches both BSA and LF through a static mechanism, as evidenced by increased UV absorption combined with fluorescence blue shift. 3.3 Molecular docking simulations Molecular docking simulations were employed to investigate the binding loci of surfactants on BSA/LF, with interaction analyses conducted in PyMOL 2.3.0 further validating the experimental findings. The final data represent the optimal docking conformation selected from multiple docking runs (Fig. 3 a-c). The optimal conformations from multiple docking runs revealed that SAP binding to BSA is primarily stabilized through hydrogen bonding interactions on the protein surface. Specifically, the carboxyl groups (-COO⁻) of negatively charged residues GLU-16 and GLU-17 in BSA formed four strong hydrogen bonds (bond lengths: 2.5 Å, 2.5 Å, 2.6 Å, 3.0 Å) with hydroxyl groups (-OH) and glycosidic oxygen atoms of SAP (Fig. 3 a). These bond lengths fall within the typical range for hydrogen bonding (2.5–3.5 Å), confirming robust interactions. Such polar interactions ensure that the sugar chains or polar groups of SAP are directionally anchored on the surface of BSA. Meanwhile, the binding of SAP may induce local conformational rearrangements, such as reduced α-helix content and increased random coil structure, affecting the exposure degree of active sites, the function of the carrier protein and the interaction with bacteria. The molecular docking results for SAP binding to LF in Fig. 3 b. Specific amino acid residues of LF engage with functional groups of saponin through multiple interactions, including two hydrogen bonds formed between LF residues and glycosidic oxygen atoms of saponin (bond lengths: 2.6 Å and 2.2 Å), complemented by van der Waals interactions involving additional residues and hydrophobic moieties of saponin. The calculated minimum binding energy of -10.82 kcal/mol indicates favorable high-affinity interactions between lactoferrin and saponin, suggesting that they can stably coexist under physiological conditions. These computational results align with experimental observations of complex saturation and conformational changes in fluorescence/UV-Vis spectra, providing atomic-level validation for stable complex formation. Molecular docking revealed significant binding characteristics between lactoferrin LF and DTAB, confirmed by a favorable binding energy of -5.2 kcal/mol, which is primarily driven by synergistic hydrogen bonding and hydrophobic forces in Fig. 3 c. The quaternary ammonium group of DTAB acts as a hydrogen bond acceptor, forming a 3.9 Å bond with LYS-637 of LF, while the hydroxyl groups of SER-519 and THR-527 in LF may serve as hydrogen bond donors, potentially forming hydrogen bonds with the bromide ion or methyl hydrogens of DTAB. These interactions collectively contribute to a stable hydrogen bonding network. Concurrently, hydrophobic interactions occur between DTAB and multiple LF residues including THR-527, TYR-524, LYS-522, GLU-521, ASP-509, LYS-520, SER-519, and GLU-353, enhancing complex stability in hydrophobic environments. The cationic quaternary ammonium group of DTAB engages in ionic bonding with negatively charged residues of LF (such as GLU-353 and ASP-509), with electrostatic forces substantially reinforcing the molecular binding. Notably, Glu-353 exhibits dual binding functionality through electrostatic attraction between its carboxyl group and quaternary ammonium headgroup of DTAB, concurrent with hydrophobic embedding via its aliphatic side chain. This polar/nonpolar dual-mode mechanism elucidates the atomic-level basis for DTAB-induced conformational changes in LF. The hydrogen bond network provides a structural basis for specific binding and rationalizes conformation alterations observed spectroscopically in LF. 3.4 Analysis of antibacterial experiment This study employed the agar diffusion method to evaluate the antibacterial properties of composite systems formed by LF or BSA with the SAP or the cationic surfactant DTAB, using E. coli and S. aureus as test organisms. The experimental setup included an LF/BSA-SAP system containing PBS control, SAP solution, LF/BSA-SAP solutions at varying concentrations and ratios, and their mixtures with DTAB; and an LF-DTAB system containing PBS control, DTAB solution, and LF-DTAB complexes at different concentrations and ratios. All samples were prepared using sterile PBS as the solvent. For the LF/BSA-SAP system, samples included (1) PBS control, (2) 50 mg/mL SAP, (3) 50 mg/mL LF/BSA-SAP (1:50), (4) 20 mg/mL LF/BSA-SAP (1:50), (5) 50 mg/mL LF/BSA-SAP (1:50) + 10 mg/mL DTAB, and (6) 20 mg/mL LF/BSA-SAP (1:50) + 10 mg/mL DTAB; for the LF-DTAB system, samples comprised (1) PBS control, (2) 50 mg/mL DTAB, (3) 50 mg/mL LF-DTAB (1:50), (4) 20 mg/mL LF-DTAB (1:50), with samples (5) and (6) both documented as 50 mg/mL LF-DTAB (1:10), indicating potential duplication in the original labeling. Experimental results revealed significant differences in antibacterial activity among the tested systems. The composite formed by the biosurfactant SAP with lactoferrin (LF-SAP, 50 mg/mL) exhibited no significant inhibitory activity against either bacterial strain (P > 0.05) in Fig. a-c. In contrast, the BSA-SAP composite demonstrated marked, concentration-dependent antibacterial effects, showing superior efficacy against S. aureus (maximum inhibition zone: 10.0 ± 0.1 mm) compared to E. coli (maximum: 5.0 ± 0.1 mm) in Fig. 4 d-f. The cationic surfactant DTAB (50 mg/mL) and its composites with LF or BSA (e.g., LF-DTAB, BSA-SAP-DTAB) exhibited potent antibacterial activity. Composites containing DTAB (≥ 20 mg/mL) achieved exceptionally strong inhibition against S. aureus (inhibition zone: 28.1 ± 1.0 mm), representing a 58.8% enhancement (P < 0.001) over the activity against E. coli . LF-DTAB composites significantly outperformed individual components (P 0.05), indicating DTAB as the key determinant of antibacterial efficacy. Proteins (LF/BSA) likely extended antimicrobial persistence through a sustained-release mechanism. The observed species-dependent antibacterial efficacy stems from structural distinctions between Gram-positive and Gram-negative bacteria. S. aureus (Gram-positive), possessing a thick, highly negatively charged peptidoglycan layer (zeta potential: -32.5 mV), exhibits enhanced susceptibility to cationic complexes such as LF-DTAB (+ 18.2 mV) via electrostatic targeting, where favorable binding energies (ΔG≈-9.8 to -10.82 kcal/mol) facilitate adsorption and membrane disruption. Conversely, E. coli (Gram-negative) demonstrates reduced susceptibility (∼36.8% lower efficacy) due to outer membrane barriers and porin size exclusion, which restricts penetration of larger complexes (hydrodynamic radius ∼4.3 nm). Synergistic mechanisms involve DTAB-mediated membrane disruption with enhanced protein permeability, electrostatic targeting by complexes, DTAB-induced conformational exposure of LF’s antimicrobial domains, iron sequestration by LF, BSA facilitating SAP enrichment and potential antimicrobial site exposure, and membrane insertion/destabilization via SAP’s hydrophobic backbone. These findings advocate for developing binary DTAB-LF/BSA formulations to harness synergistic effects, with future studies needed to optimize targeting specificity and evaluate stability in complex matrices for practical applications. 4. Conclusion This study elucidates the interaction mechanisms and functional outcomes governing the co assembly of model and functional proteins with natural and synthetic bioactive ligands. By integrating multi spectroscopic analysis with molecular docking, we decipher how specific intermolecular forces, including hydrogen bonding, hydrophobic, and electrostatic interactions, orchestrate protein conformational dynamics and complex stability. The BSA and SAP complex is stabilized primarily by cooperative hydrogen bonding and hydrophobic forces, inducing a reduction in α helical content and increasing local hydrophobicity. In contrast, LF interacts with SAP through a combination of forces without major disruption of its core hydrophobic environment. Notably, the binding of the cationic surfactant DTAB to LF, driven by synergistic hydrogen bonding, hydrophobic, and electrostatic interactions, induces significant conformational reorganization. This ligand induced remodeling directly modulates the protein’s interfacial properties and biological functionality. The antibacterial efficacy of these complexes is dictated by their colloidal‑interfacial attributes. The LF-DTAB complex displays potent, species-selective efficacy, showing superior activity against S. aureus. This selectivity arises from the DTAB-induced conformational state, which enhances the complex’s cationic character and promotes electrostatic targeting of the thick, negatively charged peptidoglycan layer in Gram-positive bacteria. The reduced activity against E. coli underscores the role of the outer‑membrane barrier in Gram‑negative organisms, highlighting the importance of matching size and surface properties in colloidal antimicrobial design. We demonstrate that controlled protein-ligand co-assembly, directed by a hierarchy of intermolecular forces, enables the reprogramming of protein conformation and the tailoring of interfacial properties to achieve enhanced bioactivity. These findings provide a molecular blueprint for engineering next-generation protein-based colloidal materials with optimized targeting and efficacy for biomedical applications. Declarations Disclosure statement The authors declare no conflict of interest. Author Contribution Yanrong Chen: conceptualization, methodology, project administration, validation, writing - original draft. Yurong Guo: investigation, formal analysis, data curation. Liwei Chen: investigation, formal analysis, data curation. Yue Shi: investigation, formal analysis. Jingwen Peng : investigation, formal analysis. Lan Bao: investigation, resources. Xing Gao: formal analysis, writing - review and editing. Suqin Han: supervision, funding acquisition, writing - review and editing. Acknowledgments This work was supported by the Natural Science Foundation of Shandong Province (ZR2024QE448), the Research Program of Qilu Institute of Technology (QIT23TP013). Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 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Ravindran A, Singh A, Raichur AM, Chandrasekaran N, Mukherjee A (2010) Studies on interaction of colloidal Ag nanoparticles with bovine serum albumin (BSA). Colloids Surf B Biointerfaces 76(1):32–37. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 22 Mar, 2026 Editor assigned by journal 04 Jan, 2026 Submission checks completed at journal 04 Jan, 2026 First submitted to journal 29 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8470788","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":610487862,"identity":"6aaee8f7-5c79-412e-a171-5092eda0cda7","order_by":0,"name":"Yanrong Chen","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanrong","middleName":"","lastName":"Chen","suffix":""},{"id":610487865,"identity":"7f96d947-971a-4006-8e5c-9970b85437f4","order_by":1,"name":"Yurong Guo","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yurong","middleName":"","lastName":"Guo","suffix":""},{"id":610487866,"identity":"a24fd394-7502-4981-b82c-5050ca806fef","order_by":2,"name":"Liwei Chen","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Liwei","middleName":"","lastName":"Chen","suffix":""},{"id":610487868,"identity":"1ed8f0ff-d642-4372-934f-b34cdc7d1b3c","order_by":3,"name":"Yue Shi","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Shi","suffix":""},{"id":610487869,"identity":"73f2e779-91bf-423a-93ee-4f0db92690c3","order_by":4,"name":"Jingwen Peng","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jingwen","middleName":"","lastName":"Peng","suffix":""},{"id":610487870,"identity":"3b67a5f2-956f-45b6-9a8c-5094d91707c1","order_by":5,"name":"Lan Bao","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Lan","middleName":"","lastName":"Bao","suffix":""},{"id":610487871,"identity":"50898199-e7af-4c5e-b4bf-bc0687d22e4d","order_by":6,"name":"Xing Gao","email":"","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Gao","suffix":""},{"id":610487872,"identity":"8b0fab3e-4098-4434-9eb5-0640016925b3","order_by":7,"name":"Suqin Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYJCCA4wNDAz8DMwNB4AcEJtILZINjA0HDhCrBazMAKSRKC3yM5I3Hi7cYZdnfDyx8fAHBhvZDQeYnz3Ap8XgRlrB4ZlnkovNzjwEOSzNeMMBNnMDvFokcgwO87YxJ267kQjScjhxwwEeNgn8DgNrqU/cPAOs5T9hLQw3wFqAhkuAtRwgrMXgzLMCoJbjiTNAfjljkGw88zCbGX6HtSdv/szbVp3Y3558+ENFhZ1s3/HmZ/gdBrQISidA2cwE1KNpGQWjYBSMglGABQAAROJW/mD8MOgAAAAASUVORK5CYII=","orcid":"","institution":"Qilu Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Suqin","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2025-12-29 08:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8470788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8470788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105383024,"identity":"e13f1b51-3652-4c89-92aa-c586bca09d13","added_by":"auto","created_at":"2026-03-25 11:42:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":130953,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The fluorescence spectrum of the BSA-SAP system. (b) The fluorescence spectrum of the LF-SAP system. (c) The fluorescence spectrum of the LF-DTAB system.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8470788/v1/41bb6c85667786a3c8e68afe.png"},{"id":105382983,"identity":"dd422fc8-9417-43fb-b204-3198c81681f6","added_by":"auto","created_at":"2026-03-25 11:42:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101930,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Absorption spectrum of BSA-SAP system. (b) Absorption spectrum of LF-SAP system. (c) Absorption spectrum of LF-DTAB system.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8470788/v1/ca0d00e78bf8839bdc3325a0.png"},{"id":105382978,"identity":"fadc1244-77f9-4799-b7c1-5bb698e84a2e","added_by":"auto","created_at":"2026-03-25 11:42:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":630562,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The molecular docking results of SAP-BSA system. (b) The molecular docking results of LF-SAP system. (c) The molecular docking results of LF-DTAB system.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8470788/v1/2e9e79bffea0a682e5e52239.png"},{"id":105382979,"identity":"471670ca-1bd4-4144-9fa8-01df2b8f17a3","added_by":"auto","created_at":"2026-03-25 11:42:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1146357,"visible":true,"origin":"","legend":"\u003cp\u003e(a-b) The antibacterial effect of LF-SAP on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. (c) The inhibition zone diameters of LF-SAP against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. (d-e) The antibacterial effect of BSA-SAP on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. (f) The inhibition zone diameters of BSA-SAP against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. (g-h) The antibacterial effect of LF-DTAB on \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. (i) The inhibition zone diameters of LF-DTAB against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8470788/v1/ddb295004a7d103a8a413e8a.png"},{"id":105565480,"identity":"c5ddd103-fa1a-4c52-9bc6-1a962eb04517","added_by":"auto","created_at":"2026-03-27 12:53:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2783008,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8470788/v1/472d025f-7234-4dc8-8ff1-39519661f4f2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Study on the Interaction Mechanisms and Antibacterial Properties of BSA/LF with SAP and LF with DTAB","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInteractions between proteins and bioactive ligands represent a cornerstone of colloid science, with profound implications for understanding biological regulation and designing advanced functional materials [1, 2]. Amid the global challenge of antimicrobial resistance, this study leverages fundamental colloidal and interfacial principles to propose a synergistic protein-ligand strategy for next-generation antimicrobials. We systematically investigate multi-scale interactions and functional modulation in three model systems: the transport protein BSA with natural SAP, the multifunctional protein LF with SAP, and LF with the cationic surfactant DTAB. This comparative design allows us to uncover general principles of molecular assembly, conformational adaptation, and functional synergy that govern bio-colloidal systems, bridging the gap between natural and synthetic bioactive hybrids.\u003c/p\u003e \u003cp\u003eThe specific scientific questions we address are threefold and interconnected. First, the complexation of SAP, a potent but poorly soluble natural biosurfactant, with the model protein BSA represents a classic colloid delivery challenge. While BSA is an established carrier [3, 4], a mechanistic and quantitative understanding of the forces driving SAP-BSA association [5, 6], the resultant protein structural dynamics, and how this translates to modulated antimicrobial efficacy remains fragmented. Second, the interaction between SAP and the multifunctional [7, 8], inherently antimicrobial protein LF is virtually unexplored. The potential for synergy between the iron-sequestration mechanism of LF [9, 10] and the membrane-disruptive action of SAP is compelling [11, 12], yet the colloidal stability, binding landscape, and functional outcome of an LF-SAP composite are unknown. Third, the interaction between LF [13, 14] and the synthetic cationic surfactant DTAB sits at the core of colloid and interface science [15, 16]. Understanding how DTAB binding remodels LF conformation, perturbs its colloidal state, and reprograms its functionality is key to designing bio-surfactant hybrids that mitigate surfactant toxicity while amplifying and diversifying antimicrobial action [17].\u003c/p\u003e \u003cp\u003eTo bridge these gaps, we employ an integrated structure-dynamics-function methodology. By combining UV-Vis and fluorescence spectroscopy with molecular docking, we decipher the hierarchy of intermolecular forces, quantify binding parameters, and map conformational transitions for each complex. Crucially, we directly correlate these molecular- and nano-scale insights with macroscopic functional performance through rigorous antibacterial assays. This work moves beyond descriptive interaction studies, establishing a colloid-science-guided framework for achieving programmable protein-ligand synergy. It provides a rational blueprint for designing next-generation antimicrobial materials, where controlled interfacial assembly and mechanistic cooperation are leveraged to overcome the limitations of single-agent systems.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eBSA (analytical grade) and LF (analytical grade) were purchased from Macklin Inc. SAP (analytical grade) and DTAB (analytical grade) were obtained from Xiya Chemical Technology Co., Ltd. Phosphate buffered saline (PBS, pH 7.4, analytical grade) was sourced from Macklin Inc. The Escherichia coli (\u003cem\u003eE. coli\u003c/em\u003e) and Staphylococcus aureus (\u003cem\u003eS. aureus\u003c/em\u003e) were acquired from Qingdao Haibo Biotechnology Co., Ltd. Peptone, yeast extract powder, and agar powder (all analytical grade) were procured from Beijing AoBoXing Bio-Tech Co., Ltd. Other analytical-grade reagents were supplied by Beijing Innochem Science \u0026amp; Technology Co., Ltd. All reagents were used without further purification. Experimental systems and controls employed pH 7.4 PBS as solvent. Glassware was rinsed with triply distilled water (conductivity\u0026thinsp;\u0026lt;\u0026thinsp;3 \u0026micro;S\u0026middot;cm⁻\u0026sup1;), and mixtures were prepared gravimetrically at predetermined ratios.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Measurement of fluorescence spectrum\u003c/h2\u003e \u003cp\u003eFluorescence spectra were acquired using an FL-4600 spectrofluorometer equipped with a thermostatic controller and 10 mm quartz cuvette. Composite solution systems were prepared by mixing protein solutions with surfactant solutions at varying concentrations. Measurements were performed at 298.2 K with excitation fixed at 280 nm and emission scanned from 300 to 500 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Measurement of UV-vis absorption spectrum\u003c/h2\u003e \u003cp\u003eUV-vis absorption spectrum of the composite solution was recorded using a UH4150 spectrophotometer equipped with a 10 mm quartz cuvette and Peltier temperature controller. Measurements were performed at 298.2 K with wavelength scanning from 200 to 350 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Molecular docking\u003c/h2\u003e \u003cp\u003eMolecular docking was performed using the following protocol: The crystal structures of BSA (PDB ID: 4F5S) and LF (PDB ID: 3IB0) were retrieved from the Protein Data Bank (PDB) [18, 19, 20]. These receptor structures were prepared with AutoDock Tools through hydrogen addition, Gasteiger charge assignment, and PDBQT format conversion [21, 22]. Ligand structures of SAP and DTAB were obtained from PubChem, followed by protonation state optimization and energy minimization using Open Babel [23]. Docking grids were centered on predicted binding sites with dimensions 40\u0026times;40\u0026times;40 \u0026Aring;. Automated docking calculations were executed in AutoDock Vina and the optimal binding pose for each complex was selected based on the lowest binding energy [24]. Protein-ligand interactions were finally visualized and analyzed using PyMOL [25].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Antibacterial testing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. Coli\u003c/em\u003e suspensions, each at a concentration of 10⁶ CFU/mL, were diluted 1000-fold in sterile phosphate-buffered saline (PBS) to obtain working suspensions [26]. Subsequently, 1 mL of each bacterial working suspension was spread evenly onto the surface of Tryptic Soy Agar (TSA) plates [27]. After allowing the inoculated plates to air-dry, sterile 6-mm-diameter filter paper discs were individually saturated with 1 \u0026micro;L of the test sample solution. The sample-saturated discs were then aseptically placed onto the inoculated TSA plates, ensuring the bacterial contact surface faced downward. Each plate was divided into six sectors: five sectors received discs saturated with test samples, while the remaining sector received a disc saturated solely with sterile PBS to serve as the negative control. All plates were incubated at 37\u0026deg;C for approximately 48 h. Following incubation, the zones of inhibition were observed, and their diameters were measured to assess antibacterial activity. The entire antibacterial assay procedure was conducted under aseptic conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Analysis of fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eTo investigate the interaction mechanisms within the composite systems, fluorescence spectroscopy was employed to monitor the influence of surfactants (SAP/DTAB) on the microenvironments surrounding amino acid residues in the proteins (BSA/LF) [28, 29]. Under conditions of fixed protein concentration, a systematic decrease in protein fluorescence emission intensity was observed across all systems (BSA-SAP, LF-SAP, LF-DTAB) with increasing concentrations of either SAP or DTAB in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This indicates a significant fluorescence quenching effect induced by the surfactants on the proteins, confirming the presence of strong interactions between them. All protein samples exhibit a characteristic fluorescence emission peak near 280 nm corresponding to the typical emission peak of tryptophan residues [30].\u003c/p\u003e \u003cp\u003eNotably, distinct patterns in λ\u003csub\u003emax\u003c/sub\u003e shifts were observed among the different systems: In the BSA-SAP system, a slight blue shift in λ\u003csub\u003emax\u003c/sub\u003e occurred with increasing SAP concentration in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. This shift suggests that the primary hydrophobic fluorophores in BSA, such as tryptophan (Trp) and tyrosine (Tyr) residues, gradually transitioned from the internal hydrophobic environment to a microenvironment of lower polarity. This directly reflects conformational rearrangements within the tertiary structure of BSA, demonstrating that SAP induces conformational changes in the protein. The stable λ\u003csub\u003emax\u003c/sub\u003e at 280 nm indicates that SAP binding does not perturb the hydrophobic microenvironment around tryptophan residues in LF in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. This suggests that the SAP-LF interaction may be strong yet non-disruptive to core structure of LF, or that the binding site is distal to these residues. For the LF-DTAB system, a blue shift in λ\u003csub\u003emax\u003c/sub\u003e was also observed as the DTAB concentration increased in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. This indicates that DTAB similarly induced alterations in the tertiary structure of LF, leading to increased exposure of its internal hydrophobic residues to a more hydrophobic microenvironment. Particularly noteworthy is that the decreasing trend in fluorescence intensity leveled off when the DTAB concentration reached 3.0\u0026times;10⁻\u0026sup2; mol/L, suggesting that the LF-DTAB complex likely reached a binding saturation state, forming a stable complex at this concentration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Anaysis of ultraviolet absorption spectrum\u003c/h2\u003e \u003cp\u003eThe UV absorption characteristics of proteins primarily arise from the π-π* electronic transitions in Trp, Tyr, and phenylalanine (Phe) residues, as well as peptide bonds [31]. Changes in UV absorption spectrum can sensitively reflect ligand-induced conformational alterations in proteins [32, 33]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, both BSA and LF exhibited characteristic absorption peaks near ~\u0026thinsp;280 nm in the absence of surfactants, attributed to the π-π* transitions of Tyr and Trp residues [34, 35]. With increasing concentrations of either SAP or DTAB, the absorbance of both proteins at 280 nm significantly increased. This indicates that the formation of surfactant-protein complexes led to the exposure of previously buried Trp/Tyr residues to the solvent environment, consequently enhancing the probability of π-π* transitions in their aromatic ring conjugated systems. This observation confirms conformational changes in the proteins.\u003c/p\u003e \u003cp\u003eFurther analysis revealed that the increasing SAP concentrations induced a slight blue shift in the maximum emission wavelength for both BSA-SAP and LF-SAP systems in both the BSA-SAP and LF-SAP systems, indicating that SAP modulates the microenvironment of aromatic amino acid residues in these proteins toward enhanced hydrophobicity and reduced polarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, while the LF-DTAB system also showed increased UV absorption, its λ\u003csub\u003eem\u003c/sub\u003e remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Static quenching involves the formation of ground-state complexes and typically alters absorption spectra, whereas dynamic quenching generally does not affect absorption characteristics. Based on this cooperative response mechanism observed in UV and fluorescence spectra, it can be inferred that SAP quenches both BSA and LF through a static mechanism, as evidenced by increased UV absorption combined with fluorescence blue shift.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Molecular docking simulations\u003c/h2\u003e \u003cp\u003eMolecular docking simulations were employed to investigate the binding loci of surfactants on BSA/LF, with interaction analyses conducted in PyMOL 2.3.0 further validating the experimental findings. The final data represent the optimal docking conformation selected from multiple docking runs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c).\u003c/p\u003e \u003cp\u003eThe optimal conformations from multiple docking runs revealed that SAP binding to BSA is primarily stabilized through hydrogen bonding interactions on the protein surface. Specifically, the carboxyl groups (-COO⁻) of negatively charged residues GLU-16 and GLU-17 in BSA formed four strong hydrogen bonds (bond lengths: 2.5 \u0026Aring;, 2.5 \u0026Aring;, 2.6 \u0026Aring;, 3.0 \u0026Aring;) with hydroxyl groups (-OH) and glycosidic oxygen atoms of SAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These bond lengths fall within the typical range for hydrogen bonding (2.5\u0026ndash;3.5 \u0026Aring;), confirming robust interactions. Such polar interactions ensure that the sugar chains or polar groups of SAP are directionally anchored on the surface of BSA. Meanwhile, the binding of SAP may induce local conformational rearrangements, such as reduced α-helix content and increased random coil structure, affecting the exposure degree of active sites, the function of the carrier protein and the interaction with bacteria.\u003c/p\u003e \u003cp\u003eThe molecular docking results for SAP binding to LF in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. Specific amino acid residues of LF engage with functional groups of saponin through multiple interactions, including two hydrogen bonds formed between LF residues and glycosidic oxygen atoms of saponin (bond lengths: 2.6 \u0026Aring; and 2.2 \u0026Aring;), complemented by van der Waals interactions involving additional residues and hydrophobic moieties of saponin. The calculated minimum binding energy of -10.82 kcal/mol indicates favorable high-affinity interactions between lactoferrin and saponin, suggesting that they can stably coexist under physiological conditions. These computational results align with experimental observations of complex saturation and conformational changes in fluorescence/UV-Vis spectra, providing atomic-level validation for stable complex formation.\u003c/p\u003e \u003cp\u003eMolecular docking revealed significant binding characteristics between lactoferrin LF and DTAB, confirmed by a favorable binding energy of -5.2 kcal/mol, which is primarily driven by synergistic hydrogen bonding and hydrophobic forces in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec. The quaternary ammonium group of DTAB acts as a hydrogen bond acceptor, forming a 3.9 \u0026Aring; bond with LYS-637 of LF, while the hydroxyl groups of SER-519 and THR-527 in LF may serve as hydrogen bond donors, potentially forming hydrogen bonds with the bromide ion or methyl hydrogens of DTAB. These interactions collectively contribute to a stable hydrogen bonding network. Concurrently, hydrophobic interactions occur between DTAB and multiple LF residues including THR-527, TYR-524, LYS-522, GLU-521, ASP-509, LYS-520, SER-519, and GLU-353, enhancing complex stability in hydrophobic environments. The cationic quaternary ammonium group of DTAB engages in ionic bonding with negatively charged residues of LF (such as GLU-353 and ASP-509), with electrostatic forces substantially reinforcing the molecular binding. Notably, Glu-353 exhibits dual binding functionality through electrostatic attraction between its carboxyl group and quaternary ammonium headgroup of DTAB, concurrent with hydrophobic embedding via its aliphatic side chain. This polar/nonpolar dual-mode mechanism elucidates the atomic-level basis for DTAB-induced conformational changes in LF. The hydrogen bond network provides a structural basis for specific binding and rationalizes conformation alterations observed spectroscopically in LF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Analysis of antibacterial experiment\u003c/h2\u003e \u003cp\u003eThis study employed the agar diffusion method to evaluate the antibacterial properties of composite systems formed by LF or BSA with the SAP or the cationic surfactant DTAB, using \u003cem\u003eE. coli\u003c/em\u003e and S. aureus as test organisms. The experimental setup included an LF/BSA-SAP system containing PBS control, SAP solution, LF/BSA-SAP solutions at varying concentrations and ratios, and their mixtures with DTAB; and an LF-DTAB system containing PBS control, DTAB solution, and LF-DTAB complexes at different concentrations and ratios. All samples were prepared using sterile PBS as the solvent. For the LF/BSA-SAP system, samples included (1) PBS control, (2) 50 mg/mL SAP, (3) 50 mg/mL LF/BSA-SAP (1:50), (4) 20 mg/mL LF/BSA-SAP (1:50), (5) 50 mg/mL LF/BSA-SAP (1:50)\u0026thinsp;+\u0026thinsp;10 mg/mL DTAB, and (6) 20 mg/mL LF/BSA-SAP (1:50)\u0026thinsp;+\u0026thinsp;10 mg/mL DTAB; for the LF-DTAB system, samples comprised (1) PBS control, (2) 50 mg/mL DTAB, (3) 50 mg/mL LF-DTAB (1:50), (4) 20 mg/mL LF-DTAB (1:50), with samples (5) and (6) both documented as 50 mg/mL LF-DTAB (1:10), indicating potential duplication in the original labeling.\u003c/p\u003e \u003cp\u003eExperimental results revealed significant differences in antibacterial activity among the tested systems. The composite formed by the biosurfactant SAP with lactoferrin (LF-SAP, 50 mg/mL) exhibited no significant inhibitory activity against either bacterial strain (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in Fig. a-c. In contrast, the BSA-SAP composite demonstrated marked, concentration-dependent antibacterial effects, showing superior efficacy against \u003cem\u003eS. aureus\u003c/em\u003e (maximum inhibition zone: 10.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mm) compared to \u003cem\u003eE. coli\u003c/em\u003e (maximum: 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 mm) in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f. The cationic surfactant DTAB (50 mg/mL) and its composites with LF or BSA (e.g., LF-DTAB, BSA-SAP-DTAB) exhibited potent antibacterial activity. Composites containing DTAB (\u0026ge;\u0026thinsp;20 mg/mL) achieved exceptionally strong inhibition against \u003cem\u003eS. aureus\u003c/em\u003e (inhibition zone: 28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 mm), representing a 58.8% enhancement (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) over the activity against \u003cem\u003eE. coli\u003c/em\u003e. LF-DTAB composites significantly outperformed individual components (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-i. Crucially, DTAB-containing composites maintained stable antibacterial activity across concentrations (20\u0026ndash;50 mg/mL) and ratios (1:10\u0026thinsp;\u0026minus;\u0026thinsp;1:50) without significant fluctuation (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating DTAB as the key determinant of antibacterial efficacy. Proteins (LF/BSA) likely extended antimicrobial persistence through a sustained-release mechanism.\u003c/p\u003e \u003cp\u003eThe observed species-dependent antibacterial efficacy stems from structural distinctions between Gram-positive and Gram-negative bacteria. \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive), possessing a thick, highly negatively charged peptidoglycan layer (zeta potential: -32.5 mV), exhibits enhanced susceptibility to cationic complexes such as LF-DTAB (+\u0026thinsp;18.2 mV) via electrostatic targeting, where favorable binding energies (ΔG\u0026asymp;-9.8 to -10.82 kcal/mol) facilitate adsorption and membrane disruption. Conversely, \u003cem\u003eE. coli\u003c/em\u003e (Gram-negative) demonstrates reduced susceptibility (\u0026sim;36.8% lower efficacy) due to outer membrane barriers and porin size exclusion, which restricts penetration of larger complexes (hydrodynamic radius \u0026sim;4.3 nm). Synergistic mechanisms involve DTAB-mediated membrane disruption with enhanced protein permeability, electrostatic targeting by complexes, DTAB-induced conformational exposure of LF\u0026rsquo;s antimicrobial domains, iron sequestration by LF, BSA facilitating SAP enrichment and potential antimicrobial site exposure, and membrane insertion/destabilization via SAP\u0026rsquo;s hydrophobic backbone. These findings advocate for developing binary DTAB-LF/BSA formulations to harness synergistic effects, with future studies needed to optimize targeting specificity and evaluate stability in complex matrices for practical applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study elucidates the interaction mechanisms and functional outcomes governing the co assembly of model and functional proteins with natural and synthetic bioactive ligands. By integrating multi spectroscopic analysis with molecular docking, we decipher how specific intermolecular forces, including hydrogen bonding, hydrophobic, and electrostatic interactions, orchestrate protein conformational dynamics and complex stability. The BSA and SAP complex is stabilized primarily by cooperative hydrogen bonding and hydrophobic forces, inducing a reduction in α helical content and increasing local hydrophobicity. In contrast, LF interacts with SAP through a combination of forces without major disruption of its core hydrophobic environment. Notably, the binding of the cationic surfactant DTAB to LF, driven by synergistic hydrogen bonding, hydrophobic, and electrostatic interactions, induces significant conformational reorganization. This ligand induced remodeling directly modulates the protein\u0026rsquo;s interfacial properties and biological functionality.\u003c/p\u003e \u003cp\u003eThe antibacterial efficacy of these complexes is dictated by their colloidal‑interfacial attributes. The LF-DTAB complex displays potent, species-selective efficacy, showing superior activity against S. aureus. This selectivity arises from the DTAB-induced conformational state, which enhances the complex\u0026rsquo;s cationic character and promotes electrostatic targeting of the thick, negatively charged peptidoglycan layer in Gram-positive bacteria. The reduced activity against E. coli underscores the role of the outer‑membrane barrier in Gram‑negative organisms, highlighting the importance of matching size and surface properties in colloidal antimicrobial design. We demonstrate that controlled protein-ligand co-assembly, directed by a hierarchy of intermolecular forces, enables the reprogramming of protein conformation and the tailoring of interfacial properties to achieve enhanced bioactivity. These findings provide a molecular blueprint for engineering next-generation protein-based colloidal materials with optimized targeting and efficacy for biomedical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYanrong Chen: conceptualization, methodology, project administration, validation, writing - original draft. Yurong Guo: investigation, formal analysis, data curation. Liwei Chen: investigation, formal analysis, data curation. Yue Shi: investigation, formal analysis. Jingwen Peng : investigation, formal analysis. Lan Bao: investigation, resources. Xing Gao: formal analysis, writing - review and editing. Suqin Han: supervision, funding acquisition, writing - review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Shandong Province (ZR2024QE448), the Research Program of Qilu Institute of Technology (QIT23TP013).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLu H, Zhou Q, He J, Jiang Z, Peng C, Tong R, Shi J (2020) Recent advances in the development of protein-protein interactions modulators: mechanisms and clinical trials. 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Colloids Surf B Biointerfaces 76(1):32\u0026ndash;37.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"colloid-and-polymer-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Colloid and Polymer Science](https://www.springer.com/journal/396) ","snPcode":"396","submissionUrl":"https://mc.manuscriptcentral.com/cps","title":"Colloid and Polymer Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Protein-bioactive interactions Antibacterial activity Molecular docking Conformational change","lastPublishedDoi":"10.21203/rs.3.rs-8470788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8470788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein-bioactive molecule interactions are fundamental to both physiological regulation and the design of advanced biofunctional systems. To address the pressing need for highly effective, low-toxicity natural antimicrobials, this work systematically examines the multi-scale interactions and antibacterial properties of model protein bovine serum albumin (BSA) and functional protein lactoferrin (LF) with natural saponin (SAP), as well as LF with the cationic surfactant dodecyltrimethylammonium bromide (DTAB). By combining UV-Vis and fluorescence spectroscopy with molecular docking, we elucidate conformation-driven structural dynamics mediated by intermolecular forces. Hydrogen bonding and hydrophobic interactions cooperatively stabilize the BSA-SAP complex, leading to a reduction in α-helical content and increased hydrophobicity around tryptophan residues. LF\u0026ndash;SAP association is governed by a combination of hydrogen bonding, van der Waals, and hydrophobic forces. Notably, DTAB binding to LF, driven by hydrogen bonding and hydrophobic interactions, induces significant conformational reorganization. Antibacterial assays demonstrate that the LF-DTAB complex exhibits strong synergistic activity against both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e, with notably enhanced efficacy against the latter. This selectivity is likely attributable to the DTAB-induced conformational state of LF and its differential affinity toward bacterial cell-wall components, possibly involving electrostatic and hydrophobic interactions. Our findings propose a protein-ligand synergy strategy to overcome limitations of single-agent antimicrobials, providing a molecular framework for the design of next-generation colloidal antimicrobial systems.\u003c/p\u003e","manuscriptTitle":"Study on the Interaction Mechanisms and Antibacterial Properties of BSA/LF with SAP and LF with DTAB","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 11:42:02","doi":"10.21203/rs.3.rs-8470788/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-03-23T03:56:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-05T04:34:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T04:32:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Colloid and Polymer Science","date":"2025-12-29T08:03:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"colloid-and-polymer-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":" Learn more about [Colloid and Polymer Science](https://www.springer.com/journal/396) ","snPcode":"396","submissionUrl":"https://mc.manuscriptcentral.com/cps","title":"Colloid and Polymer Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fd429329-8223-464f-9ba3-4e0a43ab3416","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T11:42:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 11:42:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8470788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8470788","identity":"rs-8470788","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}