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We study the dynamics of hydration water/protein association in folded proteins using lysozyme and myoglobin as examples. Extensive molecular dynamics simulations are performed to identify underlying mechanisms of the dynamical transition that corresponds to the onset of amplified atomic fluctuations in proteins. The results indicate that the number of water molecules within a cutoff distance of each residue scales linearly with protein depth index and is not affected by the local dynamics of the backbone. Keeping track of the water molecules within the cutoff sphere, we observe an effective residence time, scaling inversely with depth index at physiological temperatures while the diffusive escape is highly reduced below the transition. A depth independent orientational memory loss is obtained for the average dipole vector of the water molecules within the sphere when the protein is functional. While below the transition temperature, the solvent is in a glassy state, acting as a solid crust around the protein, inhibiting any large scale conformational fluctuations. At the transition, most of the hydration shell unfreezes and water molecules collectively make the protein more flexible. [ABSTRACT FROM AUTHOR]

Efflux-mediated β-lactam resistance is a major public health concern, reducing the effectiveness of β-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines β-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under β-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms., (© 2024. The Author(s).)

Mutational changes that affect the binding of the C2 fragment of Streptococcal protein G (GB1) to the Fc domain of human IgG (IgG-Fc) have been extensively studied using deep mutational scanning (DMS), and the binding affinity of all single mutations has been measured experimentally in the literature. To investigate the underlying molecular basis, we perform in silico mutational scanning for all possible single mutations, along with 2 μs-long molecular dynamics (WT-MD) of the wild-type (WT) GB1 in both unbound and IgG-Fc bound forms. We compute the hydrogen bonds between GB1 and IgG-Fc in WT-MD to identify the dominant hydrogen bonds for binding, which we then assess in conformations produced by Mutation and Minimization (MuMi) to explain the fitness landscape of GB1 and IgG-Fc binding. Furthermore, we analyze MuMi and WT-MD to investigate the dynamics of binding, focusing on the relative solvent accessibility of residues and the probability of residues being located at the binding interface. With these analyses, we explain the interactions between GB1 and IgG-Fc and display the structural features of binding. In sum, our findings highlight the potential of MuMi as a reliable and computationally efficient tool for predicting protein fitness landscapes, offering significant advantages over traditional methods. The methodologies and results presented in this study pave the way for improved predictive accuracy in protein stability and interaction studies, which are crucial for advancements in drug design and synthetic biology.

Antibiotic resistance presents a significant challenge to public health, as bacteria can develop resistance to antibiotics through random mutations during their life cycles, making the drugs ineffective. Understanding how these mutations contribute to drug resistance at the molecular level is crucial for designing new treatment approaches. Recent advancements in molecular biology tools have made it possible to conduct comprehensive analyses of protein mutations. Computational methods for assessing molecular fitness, such as binding energies, are not as precise as experimental techniques like deep mutational scanning. Although full atomistic alchemical free energy calculations offer the necessary precision, they are seldom used to assess high throughput data as they require significantly more computational resources. We generated a computational library using deep mutational scanning for dihydrofolate reductase (DHFR), a protein commonly studied in antibiotic resistance research. Due to resource limitations, we analyzed 33 out of 159 positions, identifying 16 single amino acid replacements. Calculations were conducted for DHFR in its drug-free state and in the presence of two different inhibitors. We demonstrate the feasibility of such calculations, made possible due to the enhancements in computational resources and their optimized use.

Genetically encoded fluorescent biosensors (GEFBs) proved to be reliable tracers for many metabolites and cellular processes. In the simplest case, a fluorescent protein (FP) is genetically fused to a sensing protein which undergoes a conformational change upon ligand binding. This drives a rearrangement in the chromophore environment and changes the spectral properties of the FP. Structural determinants of successful biosensors are revealed only in hindsight when the crystal structures of both ligand-bound and ligand-free forms are available. This makes the development of new biosensors for desired analytes a long trial-and-error process. In the current study, we conducted μs-long all atom molecular dynamics (MD) simulations of a maltose biosensor in both the apo (dark) and holo (bright) forms. We performed detailed hydrogen bond occupancy analyses to shed light on the mechanism of ligand induced conformational change in the sensor protein and its allosteric effect on the chromophore environment. We find that two strong indicators for distinguishing bright and dark states of biosensors are due to substantial changes in hydrogen bond dynamics in the system and solvent accessibility of the chromophore., (© 2024 Wiley Periodicals LLC.)

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Currently prescribed antibiotics target the catalytic sites of wild-type bacterial proteins; however, bacteria adopt mutations at this site, eventually leading to the emergence of resistance. Therefore, the identification of alternative drug binding sites is crucial, which requires knowledge of the dynamics of the mutant protein. Here, we set out to investigate the impact of a high-resistance-causing triple mutation (S385T + L389F + N526K) on the dynamics of a prioritized resistant pathogen, Haemophilus influenzae , using computational techniques. We studied penicillin-binding protein 3 (PBP3) and its complex with FtsW, which display resistance toward β-lactam antibiotics. We showed that mutations displayed local and nonlocal effects. In terms of the former, the orientation of the β-sheet, which surrounds the active site of PBP3, was impacted and the catalytic site was exposed to the periplasmic region. In addition, the flexibility of the β3-β4 loop, which modulates the catalysis of the enzyme, increased in the mutant FtsW-PBP3 complex. As for nonlocal effects, the dynamics of the pedestal domain (N-terminal periplasmic modulus (N-t)), i.e., the opening of the fork, was different between the wild-type and mutant enzymes. We showed the closed fork caused a greater number of residues to participate in the hypothesized allosteric communication network connecting N-t to the transpeptidase domain in the mutant enzyme. Finally, we demonstrated that the closed fork results in more favorable binding with β-lactam antibiotics, particularly cefixime, suggesting that small therapeutics that can stabilize the closed fork of mutant PBP3 may lead to the development of more effective molecules to combat resistant bacteria.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Prcis: The macular and peripapillary structural and vascular alterations in elderly patients with age-related choroidal atrophy may mimic glaucomatous degeneration., Purpose: To evaluate the peripapillary retinal nerve fiber layer (pRNFL), macular nerve fiber layer (mNFL), ganglion cell layer (mGCL), inner plexiform layer (mIPL) thicknesses, and choroidal vascularity index (CVI) values in patients with age-related choroidal atrophy (ARCA) and to compare with the those of age and sex-matched healthy controls., Methods: The cross-sectional, observational study included 95 eyes of 95 patients. The patients were divided into 2 groups according to subfoveal choroidal thickness (sCT) values. Forty eight eyes with sCT<125 µm constituted ARCA group (group 1), and 47 eyes with sCT>125 µm constituted control group (group 2). Peripapillary RNFL, mNFL, mGCL, mIPL, sCT values and fundus autofluorescence images were acquired with spectral domain-optical coherence tomography. Enhanced depth imaging mode spectral domain-optical coherence tomography images were binarized to show the luminal and stromal areas of choroid. CVI (%) was defined as the ratio of the luminal area to the total choroidal area with defined borders., Results: The mean age was 78.47±6.07 years in group 1 and 76.95±6.68 years in group 2. The mean, superior and inferior quadrants pRNFL thicknesses were significantly lower in group 1 than in group 2 ( P <0.05 for all). The mean mNFL, mGCL, mIPL thickness values were also statistically lower in group 1 than group 2 ( P <0.05 for all). The CVI value was 61.29±4.52 in group 1, and 64.81±3.80 in group 2 ( P <0.001)., Conclusion: The patients with ARCA had decreased pRNFL, inner macular layer thickness and CVI values when compared with healthy control eyes. These findings should be considered in the differential diagnosis and the follow up of eyes with glaucoma., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: The use of biological agents in the treatment of ocular Behçet's disease has recently become more frequent. The use of two agents, infliximab (IFX) and adalimumab (ADA), for the treatment of Behçet's disease requires prior approval by the Turkish Medicines and Medical Devices Agency. We report on a review of such applications with a view to informing on how such agents are used off-label in Turkey., Methods: Prescriptions for off-label use of IFX or ADA sent from hospitals in Turkey to the Turkish Medicines and Medical Devices Agency in 2018 were evaluated. Demographic data, previous treatment regimens and reasons for referral were extracted from the files of the cases., Results: A total of 662 patients were considered for off-label use of IFX or ADA for the treatment of ocular Behçet's disease. The mean age of the patients was 35.7±10.8 years (range 12-76); 61.5% of patients were men and 38.5% were women. Of the applications, 345 (52.1%) were for IFX and 317 (47.9%) for ADA. Among the referring hospitals, the public university hospitals ranked first, accounting for 77.9% of IFX and 88.6% of ADA prescriptions. Most applications were made after the failure of conventional therapy, which included steroids and immunosuppressive agents., Conclusion: IFX and ADA are rarely used as initial therapy. Stepwise treatment is still preferred in the treatment of ocular Behçet's disease in Turkey. Our report informs on the management of this difficult-to-treat condition., Competing Interests: Competing interests: None declared., (© European Association of Hospital Pharmacists 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Antibiotic resistance is a global health problem in which mutations occurring in functional proteins render drugs ineffective. The working mechanisms of the arising mutants are seldom apparent; a methodology to decipher these mechanisms systematically would render devising therapies to control the arising mutational pathways possible. Here we utilize C α -C β bond vector relaxations obtained from moderate length MD trajectories to determine conduits for functionality of the resistance conferring mutants of Escherichia coli dihydrofolate reductase. We find that the whole enzyme is synchronized to the motions of the substrate, irrespective of the mutation introducing gain-of-function or loss-of function. The total coordination of the motions suggests changes in the hydrogen bond dynamics with respect to the wild type as a possible route to determine and classify the mode-of-action of individual mutants. As a result, nine trimethoprim-resistant point mutations arising frequently in evolution experiments are categorized. One group of mutants that display the largest occurrence (L28R, W30G) work directly by modifying the dihydrofolate binding region. Conversely, W30R works indirectly by the formation of the E139-R30 salt bridge which releases energy resulting from tight binding by distorting the binding cavity. A third group (D27E, F153S, I94L) arising as single, resistance invoking mutants in evolution experiment trajectories allosterically and dynamically affects a hydrogen bonding motif formed at residues 59-69-71 which in turn modifies the binding site dynamics. The final group (I5F, A26T, R98P) consists of those mutants that have properties most similar to the wild type; these only appear after one of the other mutants is fixed on the protein structure and therefore display clear epistasis. Thus, we show that the binding event is governed by the entire enzyme dynamics while the binding site residues play gating roles. The adjustments made in the total enzyme in response to point mutations are what make quantifying and pinpointing their effect a hard problem. Here, we show that hydrogen bond dynamics recorded on sub-μs time scales provide the necessary fingerprints to decipher the various mechanisms at play.

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRAS WT and aim to invoke similar behavior in HRAS G12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRAS G12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRAS G12D /RAF interaction was prevented by the ligand in HEK293T cells that expressed HRAS G12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins., Competing Interests: MI, RK, FJ, CA, OT, NK, OS No competing interests declared, (© 2022, Ilter, Kasmer et al.)

Human serum transferrin binds ferric ions with high affinity and delivers them into cells via receptor-mediated endocytosis upon a decrease in pH in the endosome. Protonation events and conformational changes are known to play an important role in iron-release though the release is not yet fully understood. Human serum transferrin consists of two similar lobes which release iron at different rates. In this study, we investigate the iron binding sites of N- and C-lobes using quantum mechanical tools, particularly, the quantum chemical cluster approach. This study supports the inevitable role of axial tyrosine for the release of iron in quantum chemical models and provides valuable information about the proton transfer pathways for the protonation of Tyr188 and Tyr517 in N- and C-lobes, respectively. The calculations show that the release process is similar in both lobes; however, the energetic differences of the release process in N- and C-lobes, demonstrated for the first time, indicated that the release of iron in the N-lobe is thermodynamically favorable, in contrast to the one in the C-lobe.

The BTB domain is an oligomerization domain found in over 300 proteins encoded in the human genome. In the family of BTB domain and zinc finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site, whereas the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study, we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed cell-based binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo- and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family., (© 2022 Piepoli et al.)

Protein function is constrained by the three-dimensional structure but is delineated by its dynamics. This framework must satisfy specificity of function along with adaptability to changing environments and evolvability under external constraints. The accessibility of the available regions of the energy landscape for a set of conditions and shifts in the populations upon their modulation have effects propagating across scales, from biomolecular interactions, to organisms, to populations. Developing the ability to detect and juggle protein conformations supplemented by a physics-based understanding has implications for not only in vivo problems but also for resistance impeding drug discovery and bionano-sensor design., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: This cross-sectional study compared the peripapillary vessel density and retinal nerve fiber layer (RNFL) thickness in patients with exfoliation syndrome (XFS) and healthy controls for evaluation of the early structural and vascular alterations in XFS., Methods: One eye was included from 75 patients with XFS and 54 healthy controls. The patients with XFS were matched the controls for age, intraocular pressure and axial length. The vascular density of the radial peripapillary capillaries (RPCs) and the peripapillary RNFL thickness were evaluated with optical coherence tomography angiography., Results: The mean peripapillary RNFL thicknesses of the groups were similar in all sectors (p > 0.05 for all). However, eyes with XFS demonstrated lower mean peripapillary vessel densities in all areas (p < 0.05 for all) except for the nasal sector (p = 0.68) compared to the controls. The gradual age correlated decline in the peripapillary RNFL thickness and the RPC vessel density observed in the healthy eyes was absent in XFS (r = - 0.14 p = 0.65 and r = - 0.23 p = 0.05)., Conclusions: Alterations in the peripapillary vascular density despite a preserved RNFL thickness in XFS supports the hypothesis that vascular alterations may precede structural alterations and have an important role in the pathogenesis of XFS. XFS may have different effects on the microvasculature of different peripapillary areas, with the nasal sector being mostly preserved., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Purpose: To investigate the effect of microalbuminuria (MA) on superficial vessel density (SVD), deep vessel density (DVD), and choriocapillaris vessel density (CVD) in type-2 diabetic patients., Methods: Twenty patients without diabetic retinopathy (DR) and MA (group 1), 20 patients without DR but with MA (group 2) and 30 patients with mild DR and MA (group 3) were enrolled in this prospective and cross-sectional study. SVD, DVD, and CVD of all patients were screened with optical coherence tomography angiography (OCTA). The relationships between these values and age, diabetes duration and metabolic parameters were also evaluated., Results: The whole macular SVD value was 50.15 ± 4.52 in group 1 and 47.81 ± 4.12 in group 2 ( p = .04). The whole macular DVD value was 47.66 ± 2.76 in group 1, 44.37 ± 3.39 in group 3 ( p = .02). Parafoveal DVD value was 52.58 ± 3.47 in group 1, 51.84 ± 2.23 in group 2, and 49.23 ± 3.38 in group 3 (p G1&3 = .001, p G2&3 = .02). Perifoveal DVD value was 47.92 ± 3.30 in group 1, 43.96 ± 4.19 in group 2, and 42.85 ± 2.98 in group 3 (p G1&2 = .02 and p G1&3 < .001). There were inverse correlations between diabetes duration, urea, creatinine, albumin, urinary sodium and some DVD values ( p < .05, for all). Also, there were inverse correlations between parafoveal and perifoveal DVD values and MA ( p = .002 and p = .031). Additionally, inverse correlations were determined between diabetes duration, creatinine, urea, serum Na and some CVD values ( p < .05 for all). Conclusion : Decreased SVD and DVD values measured by OCTA in type-2 diabetic patients, whether they have mild DR or not, may be associated with MA causing early retinal microvascular changes.

Purpose: To determine the demographic and clinical characteristics of newborn patients who underwent lacrimal probing surgical intervention with or without the marsupialization of intranasal cysts as the primary management for dacryocystocele treatment., Methods: Data from the medical charts of 350 infants who underwent lacrimal probing surgery due to nasolacrimal duct obstruction were reviewed retrospectively. Ten newborn patients with a naive diagnosis of congenital dacryocystocele were included in the study. Congenital dacryocystocele diagnosis was based on a triad of swelling in the inner canthal region, a bluish appearance, and epiphora. Lacrimal probing surgery accompanied by nasal endoscopy was planned for all patients as the first treatment option., Results: The mean age of the patients was 24.90 ± 7.15 days, with a range of 6-85 days. A total of 10 patients were included, comprising seven females and three males. The mean postoperative follow-up period was 38.7 ± 24.41 months. Five patients had left, four patients had right, and one patient had bilateral dacryocystocele. Seven eyes of the six patients had uncomplicated dacryocystocele, while the remaining patients had dacryocystocele with complications of dacryocystitis and/or preseptal cellulitis. All patients had intranasal cysts. All patients underwent one session of lacrimal probing surgery under general anesthesia; all with successful outcomes. Four patients with additional dacryocystocele-associated complications underwent combined intranasal marsupialization of the cyst wall., Discussion: Lacrimal probing surgery ± intranasal marsupialization of the cyst wall as a first treatment option can be effective for both congenital dacryocystocele and/or congenital dacryocystocele plus associated complications and provide complete resolution of dacryocystocele-related symptoms., Competing Interests: Conflict of interest statement: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2021.)

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.

The third domain of PSD-95 (PDZ3) is a model for investigating allosteric communication in protein and ligand interactions. While motifs contributing to its binding specificity have been scrutinized, a conformational dynamical basis is yet to be established. Despite the miniscule structural changes due to point mutants, the observed significant binding affinity differences have previously been assessed with a focus on two α-helices located at the binding groove (α 2 ) and the C-terminus (α 3 ). Here, we employ a new computational approach to develop a generalized view on the molecular basis of PDZ3 binding selectivity and interaction communication for a set of point mutants of the protein (G330T, H372A, G330T-H372A) and its ligand (CRIPT, named L 1 , and its T-2F variant, L 2 ) along with the wild type (WT). To analyze the dynamical aspects hidden in the conformations that are produced by molecular dynamics simulations, we utilize variations in community composition calculated based on the betweenness centrality measure from graph theory. We find that the highly charged N-terminus, which is located far from the ligand, has the propensity to share the same community with the ligand in the biologically functional complexes, indicating a distal segment might mediate the binding dynamics. N- and C-termini of PDZ3 share communities, and α 3 acts as a hub for the whole protein by sustaining the communication with all structural segments, albeit being a trait not unique to the functional complexes. Moreover, α 2 which lines the binding cavity frequently parts communities with the ligand and is not a controller of the binding but is rather a slave to the overall dynamics coordinated by the N-terminus. Thus, ligand binding fate in PDZ3 is traced to the population of community compositions extracted from dynamics despite the lack of significant conformational changes.

PDZ domains constitute common models to study single-domain allostery without significant structural changes. The third PDZ domain of PSD-95 (PDZ3) is known to have selective structural features that confer unique modulatory roles to this unit. In this model system, two residues, H372 directly connected to the binding site and G330 holding an off-binding-site position, were designated to assess the effect of mutations on binding selectivity. It has been observed that the H372A and G330T-H372A mutations change ligand preferences from class I (T/S amino acid at position -2 of the ligand) to class II (hydrophobic amino acid at the same position). Alternatively, the G330T single mutation leads to the recognition of both ligand classes. We have performed a series of molecular dynamics (MD) simulations for wild-type, H372A, and G330T single mutants and a double mutant of PDZ3 in the absence and presence of both types of ligands. With the combination of free-energy difference calculations and a detailed analysis of MD trajectories, "class switching" and "class bridging" behavior of PDZ3 mutants, as well as their effects on ligand selection and binding affinities are explained. We show that the dynamics of the charged N-terminus plays a fundamental role in determining the binding preferences in PDZ3 by altering the electrostatic energy. These findings are corroborated by simulations on N-terminus-truncated versions of these systems. The dynamical allostery orchestrated by the N-terminus offers a fresh perspective to the study of communication pathways in proteins.

Purpose: To evaluate the anatomic changes in the cornea and anterior segment following intravitreal aflibercept loading dose for neovascular age-related macular degeneration., Methods: The study included 40 eyes of 40 patients with neovascular age-related macular degeneration. Each patient underwent a loading dose of one injection per month for three consecutive doses of aflibercept (0.05 ml/2 mg). Before and after the loading dose, a record was made for each patient of corneal topography, anterior segment, corneal densitometry, and lens densitometry parameters with the Pentacam HR and specular microscopy parameters with a non-contact specular microscope. The data before and after the aflibercept loading dose were compared., Results: Corneal densitometry parameters in the 0- to 2-mm and 2- to 6-mm concentric zones of the posterior layer were significantly higher after the loading dose compared with baseline ( p  = 0.03, p  = 0.04, respectively). Corneal densitometry parameters of the anterior, central, and total corneal layer in the 10- to 12-mm concentric zone were also significantly higher after the loading dose compared with baseline ( p  = 0.009, p  = 0.02, and p  = 0.007, respectively). No significant changes were determined in respect of corneal topography, anterior segment, lens densitometry, and specular microscopy parameters ( p  > 0.05 for all)., Conclusion: The aflibercept loading dose caused slightly increased densitometric values in some corneal regions while it did not affect the corneal topography, anterior segment, lens densitometry, and specular microscopy parameters., Competing Interests: Conflict of interest statement: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s), 2020.)

We address the problem of triggering dissociation events between proteins that have formed a complex. We have collected a set of 25 non-redundant, functionally diverse protein complexes having high-resolution three-dimensional structures in both the unbound and bound forms. We unify elastic network models with perturbation response scanning (PRS) methodology as an efficient approach for predicting residues that have the propensity to trigger dissociation of an interacting protein pair, using the three-dimensional structures of the bound and unbound proteins as input. PRS reveals that while for a group of protein pairs, residues involved in the conformational shifts are confined to regions with large motions, there are others where they originate from parts of the protein unaffected structurally by binding. Strikingly, only a few of the complexes have interface residues responsible for dissociation. We find two main modes of response: In one mode, remote control of dissociation in which disruption of the electrostatic potential distribution along protein surfaces play the major role; in the alternative mode, mechanical control of dissociation by remote residues prevail. In the former, dissociation is triggered by changes in the local environment of the protein, e.g., pH or ionic strength, while in the latter, specific perturbations arriving at the controlling residues, e.g., via binding to a third interacting partner is required for decomplexation. We resolve the observations by relying on an electromechanical coupling model which reduces to the usual elastic network result in the limit of the lack of coupling. We validate the approach by illustrating the biological significance of top residues selected by PRS on select cases where we show that the residues whose perturbation leads to the observed conformational changes correspond to either functionally important or highly conserved residues in the complex., (Copyright © 2020 Abdizadeh, Jalalypour, Atilgan and Atilgan.)

Conformational transitions in proteins facilitate precise physiological functions. Therefore, it is crucial to understand the mechanisms underlying these processes to modulate protein function. Yet, studying structural and dynamical properties of proteins is notoriously challenging due to the complexity of the underlying potential energy surfaces (PES). We have previously developed the perturbation-response scanning (PRS) method to identify key residues that participate in the communication network responsible for specific conformational transitions. PRS is based on a residue-by-residue scan of the protein to determine the subset of residues/forces which provide the closest conformational change leading to a target conformational state, inasmuch as linear response theory applies to these motions. Here, we develop a novel method to further evaluate if conformational transitions may be triggered on the PES. We aim to study functionally relevant conformational transitions in proteins by using results obtained from PRS and feeding them as inputs to steered molecular dynamics simulations. The success and the transferability of the method are evaluated on three protein systems having different complexities of motion on the PES: calmodulin, adenylate kinase, and bacterial ferric binding protein. We find that the method captures the target conformation, while providing key residues and the optimum paths with relatively low free energy profiles.

PATZ1 is a ubiquitously expressed transcriptional repressor belonging to the ZBTB family that is functionally expressed in T lymphocytes. PATZ1 targets the CD8 gene in lymphocyte development and interacts with the p53 protein to control genes that are important in proliferation and in the DNA-damage response. PATZ1 exerts its activity through an N-terminal BTB domain that mediates dimerization and co-repressor interactions and a C-terminal zinc-finger motif-containing domain that mediates DNA binding. Here, the crystal structures of the murine and zebrafish PATZ1 BTB domains are reported at 2.3 and 1.8 Å resolution, respectively. The structures revealed that the PATZ1 BTB domain forms a stable homodimer with a lateral surface groove, as in other ZBTB structures. Analysis of the lateral groove revealed a large acidic patch in this region, which contrasts with the previously resolved basic co-repressor binding interface of BCL6. A large 30-amino-acid glycine- and alanine-rich central loop, which is unique to mammalian PATZ1 amongst all ZBTB proteins, could not be resolved, probably owing to its flexibility. Molecular-dynamics simulations suggest a contribution of this loop to modulation of the mammalian BTB dimerization interface., (open access.)

Purpose: To investigate the effect of microalbuminuria on macular thickness in patients with type-2 diabetes mellitus with no or mild diabetic retinopathy and to investigate the relationship between macular thickness and metabolic parameters., Materials and Methods: Fifty eight eyes of 58 patients without diabetic retinopathy (group 1) in microalbuminuria stage, 42 eyes of 42 patients with mild diabetic retinopathy (group 2) in microalbuminuria stage, and 50 eyes of 50 patients without diabetic retinopathy and microalbuminuria (group 3) were included in this study. After detailed ophthalmologic examination, all patients underwent spectral domain-optical coherence tomography measurements. Macular thickness was noted from nine different areas (fovea, four parafoveal, and four perifoveal areas) and compared between groups. The correlations between macular thickness and age, duration of diabetes mellitus, microalbuminuria, serum urea, creatinine, glycosylated hemoglobin (HbAIc), albumin, sodium (Na), and urinary Na were evaluated., Results: The mean age was 53.29 ± 6.49 in group 1, 55.86 ± 6.97 in group 2, and 52.98 ± 5.66 years in group 3 (p = 0.06). The macular thickness values of superior, inferior, and nasal parafoveal areas were significantly different between groups (p = 0.001, p = 0.006, and p = 0.03, respectively). Bonferroni post test revealed that this difference originated from the difference between group 2 and 3 (p < 0.05 for all values). There were significant negative correlations between the macular thickness values of parafoveal areas and serum urea, HbA1c, albumin, microalbuminuria levels (p < 0.05 for all values)., Conclusion: In this study, a significantly decreased parafoveal macular thickness was measured in patients with mild diabetic retinopathy and microalbuminuria compared to patients without diabetic retinopathy and microalbuminuria.

Evolutionary fitness landscapes of several antibiotic target proteins have been comprehensively mapped showing strong high-order epistasis between mutations, but understanding these effects at the biochemical and structural levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics of Escherichia coli dihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim-induced selection. To facilitate this, we developed a new in vitro assay for rapidly characterizing DHFR steady-state kinetics. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct changes in the catalytic efficiencies of mutated DHFR enzymes. Next, we measured biochemical parameters (Km, Ki, and kcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the high-order epistasis in catalytic power of DHFR (kcat and Km) creates a rugged fitness landscape under trimethoprim selection. Taken together, our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Thermoplastic polyurethanes (TPUs) are designed using a large variety of basic building blocks but are only synthesized in a limited number of solvent systems. Understanding the behavior of the copolymers in a selected solvent system is of particular interest to tune the intricate balance of microphase separation/mixing, which is the key mechanism behind the structure formation in TPUs. Here, we present a computationally efficient approach for selecting TPU building blocks and solvents based on their Flory-Huggins interaction parameters for a precise control over the microphase separation/mixing. We first cluster eight soft segments (PEO, PPO, PTMO, PBA, PCL, PDMS, PIB, or PEB) used frequently in TPUs into three categories according to the strength of their interactions with the binary solvent THF/DMF. We then perform a comprehensive set of dissipative particle dynamics simulations of the TPUs in a range of solvent ratios. This enables us to demonstrate the emergence of the unusual channel-like structures in a narrow range of parameters and to determine the critical interactions operative for obtaining either microphase separated or mixed structures. The findings are supported by thermodynamic arguments. The approach developed here is useful for designing novel TPUs with well-defined conformational characteristics, controlled morphologies, and advanced functional properties.

Because of the serious neurologic consequences of iron deficiency and iron excess in the brain, interest in the iron status of the central nervous system has increased significantly in the past decade. While iron plays an important role in many physiological processes, its accumulation may lead to diseases such as Huntington's, Parkinson's, and Alzheimer's. Therefore, it is important to develop methodologies that can monitor the presence of iron in a selective and sensitive manner. In this paper, we first showed the synthesis and characterization of the iron-binding protein (FBP) from Haemophilus influenzae, specific for ferrous ions. Subsequently, we employed this protein in our nanopipette platform and utilized it in functionalized nanoprobes to monitor the presence of ferrous ions. A suite of characterization techniques: absorbance spectroscopy, dynamic light scattering, and small-angle X-ray scattering were used for FBP. The functionalized Fe-nanoprobe calibrated in ferrous chloride enabled detection from 0.05 to 10 μM, and the specificity of the modified iron probe was evaluated by using various metal ion solutions.

Purpose: To evaluate the corneal tomographic parameters in patients with familial Mediterranean fever (FMF) and to compare data with those of healthy control subjects., Materials and Methods: Forty eyes of 40 patients with FMF (FMF group) and 35 eyes of 35 healthy subjects (control group) were included to this prospective study. All participants underwent complete ophthalmological examination and tomographic analysis with Pentacam HR. Maximum keratometry, front and back astigmatism, thinnest pachymetry, maximum anterior and posterior elevation best-fit-sphere (BFS) at the 5.0 mm zone, front and back difference elevation, final D, average and maximum pachymetric progression index, and maximum Ambrósio relational thickness parameters were noted., Results: Groups were similar in terms of age and gender (p = 0.77 and 0.35). Maximum keratometry, thinnest pachymetry, front and back astigmatism, and maximum anterior elevation BFS at the 5.0 mm values were similar between groups (p = 0.22, 0.52, 0.49, 0.29, and 0.31, respectively). Maximum posterior elevation BFS at the 5.0 mm and back difference elevation values were higher than control group in FMF group (p = 0.001 and 0.04). The mean values of final D, average and maximum pachymetric progression index were higher, and Ambrósio relational thickness was lower in FMF group compared to control group (p = 0.03, 0.002, 0.006, and 0.01, respectively)., Conclusions: FMF patients carry increased corneal ectasia risk compared to healthy subjects. The corneal tomographic characteristics and the association of keratoconus with FMF may be important in a detailed refractive surgery work-up.

Summary: MODE-TASK, a novel and versatile software suite, comprises Principal Component Analysis, Multidimensional Scaling, and t-Distributed Stochastic Neighbor Embedding techniques using Molecular Dynamics trajectories. MODE-TASK also includes a Normal Mode Analysis tool based on Anisotropic Network Model so as to provide a variety of ways to analyse and compare large-scale motions of protein complexes for which long MD simulations are prohibitive. Beside the command line function, a GUI has been developed as a PyMOL plugin., Availability and Implementation: MODE-TASK is open source, and available for download from https://github.com/RUBi-ZA/MODE-TASK. It is implemented in Python and C++. It is compatible with Python 2.x and Python 3.x and can be installed by Conda., Supplementary Information: Supplementary data are available at Bioinformatics online.

Enterovirus 71 can be a severe pathogen in small children and immunocompromised adults. Virus uncoating is a critical step in the infection of the host cell; however, the mechanisms that control this process remain poorly understood. We applied normal mode analysis and perturbation response scanning to several complexes of the virus capsid and present a coarse-graining approach to analyze the full capsid. We show that our method offers an alternative to expressing the system as a set of rigid blocks and accounts for the interconnection between nodes within each subunit and protein interfaces across the capsid. In our coarse-grained approach, the modes associated with capsid expansion are captured in the first three nondegenerate modes and correspond to the changes observed in structural studies of the virus. We show that the resolution of the analysis may be modified without losing information on the global motions leading to uncoating. Perturbation response scanning revealed that a protomer cannot serve as a functional unit to explain deformations of the capsid. Instead, we define a pentamer as the minimum functional unit to investigate changes within the capsid. From the modal analysis and perturbation response scanning, we locate a hotspot region surrounding the fivefold axis. The range of the effect of these single, hotspot residues extend to 140 Å. The perturbation of internal capsid residues in this region displayed greatest propensity to capsid expansion, thus indicating the significant role that the RNA genome may play in triggering uncoating., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Central to Hsp90's biological function is its ability to interconvert between various conformational states. Drug targeting of Hsp90's regulatory mechanisms, including its modulation by cochaperone association, presents as an attractive therapeutic strategy for Hsp90 associated pathologies. In this study, we utilized homology modeling techniques to calculate full-length structures of human Hsp90α in closed and partially open conformations and used these structures as a basis for several molecular dynamics based analyses aimed at elucidating allosteric mechanisms and modulation sites in human Hsp90α. Atomistic simulations demonstrated that bound adenosine triphosphate (ATP) stabilizes the dimer by "tensing" each protomer, while adenosine diphosphate (ADP) and apo configurations "relax" the complex by increasing global flexibility, the former case resulting in a fully open "v-like" conformation. Dynamic residue network analysis revealed regions of the protein involved in intraprotein communication and identified several key communication hubs that correlate with known functional sites. Pairwise comparison of betweenness centrality, shortest path, and residue fluctuations revealed that a proportional relationship exists between the latter two measurables and an inverse relationship between these two and betweenness centrality. This analysis showed how protein flexibility, degree of compactness, and the distance cutoff used for network construction influence the correlations between these metrics. These findings are novel and suggest shortest path and betweenness centrality to be more relevant quantities to follow for detecting functional residues in proteins compared to residue fluctuations. Perturbation response scanning analysis identified several potential residue sites capable of modulating conformational change in favor of interstate conversion. For the ATP-bound open conformation, these sites were found to overlap with known Aha1 and client binding sites, demonstrating how naturally occurring forces associated with cofactor binding could allosterically modulate conformational dynamics.

Methods developed toward computational exploration of protein landscapes have become standardized tools to assess biophysical experimental findings. They are also used on their own right to discover the workings of the protein as a molecular machine, potential sites of interest for protein functioning, allosteric regions in proteins, and communication pathways between different sites on a protein. With the development of reliable force fields that describe interactions in biomolecules, molecular dynamics (MD) simulations have become the prime tool for this purpose. While it is now straightforward to carry out MD simulations up to microseconds with current computers readily available to researchers, many processes of biological interest occur on several of orders of magnitudes slower timescales. Thus, the latter problems are attackable through MD by a handful of researchers that have access to the most powerful computers. Alternatively, physics-based methods to interrogate the protein energy landscape are in continuous development to circumvent this problem. In addition to opening the routes for advancement to a large number of researchers that have access to modest computational resources, they have the advantage of providing an understanding of the mechanisms that govern protein dynamics. Here we discuss network-based approaches geared toward understanding protein dynamics. These include (i) construction of residue networks which view proteins as networks of nodes connected through local interactions and (ii) construction of proteins as elastic networks whose modes of motion may be manipulated to achieve allowed conformational changes. Limitations of the methods as well as opportunities for future exploitation are described., (© 2018 Elsevier Inc. All rights reserved.)

In this prospective study, the biomechanical properties of optic nerve head (ONH) and cornea in both eyes of patients with non-arteritic anterior ischaemic optic neuropathy and healthy control eyes were investigated. ONH elastometry was measured with real-time elastography, and corneal elastometry was measured with ocular response analyser. Elastometry of cornea and ONH was lower in both eyes of patients with unilateral non-arteritic ischaemic optic neuropathy than in healthy control eyes. The role of these biomechanical differences in the pathogenesis of non-arteritic ischaemic optic neuropathy should be investigated further.

Summary: Molecular dynamics (MD) determines the physical motions of atoms of a biological macromolecule in a cell-like environment and is an important method in structural bioinformatics. Traditionally, measurements such as root mean square deviation, root mean square fluctuation, radius of gyration, and various energy measures have been used to analyze MD simulations. Here, we present MD-TASK, a novel software suite that employs graph theory techniques, perturbation response scanning, and dynamic cross-correlation to provide unique ways for analyzing MD trajectories., Availability and Implementation: MD-TASK has been open-sourced and is available for download from https://github.com/RUBi-ZA/MD-TASK , implemented in Python and supported on Linux/Unix., Contact: o.tastanbishop@ru.ac.za., (© The Author(s) 2017. Published by Oxford University Press.)

Hsp70 molecular chaperones play an important role in maintaining cellular homeostasis, and are implicated in a wide array of cellular processes, including protein recovery from aggregates, cross-membrane protein translocation, and protein biogenesis. Hsp70 consists of two domains, a nucleotide binding domain (NBD) and a substrate binding domain (SBD), each of which communicates via an allosteric mechanism such that the protein interconverts between two functional states, an ATP-bound open conformation and an ADP-bound closed conformation. The exact mechanism for interstate conversion is not as yet fully understood. However, the ligand-bound states of the NBD and SBD as well as interactions with cochaperones such as DnaJ and nucleotide exchange factor are thought to play crucial regulatory roles. In this study, we apply the perturbation-response scanning (PRS) method in combination with molecular dynamics simulations as a computational tool for the identification of allosteric hot residues in the large multidomain Hsp70 protein. We find evidence in support of the hypothesis that substrate binding triggers ATP hydrolysis and that the ADP-substrate complex favors interstate conversion to the closed state. Furthermore, our data are in agreement with the proposal that there is an allosterically active intermediate state between the open and closed states and vice versa, as we find evidence that ATP binding to the closed structure and peptide binding to the open structure allosterically "activate" the respective complexes. We conclude our analysis by showing how our PRS data fit the current opinion on the Hsp70 conformational cycle and present several allosteric hot residues that may provide a platform for further studies to gain additional insight into Hsp70 allostery.

The dynamical and thermodynamic behavior of human transferrin (hTf) protein in saline aqueous solution of various concentrations is studied. hTf is an essential transport protein circulating iron in the blood and delivering it to tissues. It displays highly pH dependent cooperativity between the two lobes, each carrying an iron, and forms a tight complex with the receptor during endocytosis, eventually recycled to the serum after iron release. Molecular dynamics simulations are used to investigate the effects of the amount of salt on protein conformation and dynamics to analyze the structure-function relationship in free hTf at serum pH. To monitor the ionic strength dependence, four different ionic concentrations, 0, 50, 130, and 210 mM NaCl for two protonation states of the iron coordination site is considered. Two mechanisms by which salt affects hTf are disclosed. In the totally closed state where iron coordinating tyrosines are deprotonated, the addition of even 50 mM of salt alters the electrostatic potential distribution around the protein, opening energetic pathways for tyrosine protonation from nearby charged residues as a required first step for iron release. Once domain opening is observed, conformational plasticity renders the iron binding site more accessible by the solvent. At this second stage of iron release, R124 in the N-lobe is identified as kinetically significant anion binding site that accommodates chloride ions and allosterically communicates with the iron binding residues. Opening motions are maximized at 150 mM IS in the N-lobe, and at 210 mM in the C-lobe. The extra mobility in the latter is thought to preclude binding of hTf to its receptor. Thus, the physiological IS is optimal for exposing iron for release from hTf. However, the calculated binding affinities of iron show that even in the most open conformations, iron dissociation needs to be accompanied by chelators.

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme with an essential role in cell metabolism. DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is a precursor for purine and thymidylate synthesis. Several DHFR targeting antifolate drugs including trimethoprim, a competitive antibacterial inhibitor, have therefore been developed and are clinically used. Evolution of resistance against antifolates is a common public health problem rendering these drugs ineffective. To combat the resistance problem, it is important to understand resistance-conferring changes in the DHFR structure and accordingly develop alternative strategies. Here, we structurally and dynamically characterize Escherichia coli DHFR in its wild type (WT) and trimethoprim resistant L28R mutant forms in the presence of the substrate and its inhibitor trimethoprim. We use molecular dynamics simulations to determine the conformational space, loop dynamics and hydrogen bond distributions at the active site of DHFR for the WT and the L28R mutant. We also report their experimental kcat, Km, and Ki values, accompanied by isothermal titration calorimetry measurements of DHFR that distinguish enthalpic and entropic contributions to trimethoprim binding. Although mutations that confer resistance to competitive inhibitors typically make enzymes more promiscuous and decrease affinity to both the substrate and the inhibitor, strikingly, we find that the L28R mutant has a unique resistance mechanism. While the binding affinity differences between the WT and the mutant for the inhibitor and the substrate are small, the newly formed extra hydrogen bonds with the aminobenzoyl glutamate tail of DHF in the L28R mutant leads to increased barriers for the dissociation of the substrate and the product. Therefore, the L28R mutant indirectly gains resistance by enjoying prolonged binding times in the enzyme-substrate complex. While this also leads to slower product release and decreases the catalytic rate of the L28R mutant, the overall effect is the maintenance of a sufficient product formation rate. Finally, the experimental and computational analyses together reveal the changes that occur in the energetic landscape of DHFR upon the resistance-conferring L28R mutation. We show that the negative entropy associated with the binding of trimethoprim in WT DHFR is due to water organization at the binding interface. Our study lays the framework to study structural changes in other trimethoprim resistant DHFR mutants.

Ferric binding protein (FbpA) is part of an elaborate iron piracy mechanism evolved in Gram-negative bacteria, shuttling iron in the periplasmic space, from the outer to the cytoplasmic membrane side. We address how the dissociation process of iron is facilitated, since the binding constant of iron is on the order of 10 18 M -1 at 6.5 pH and 200 mM ionic strength (IS). We monitor the conformational preferences of FbpA by extensive molecular dynamics (MD) simulations under conditions where IS, charge states of iron coordinating tyrosines and pH are varied, as well as when a mutation is introduced at an allosteric site. Steered MD is utilized to predict the binding affinity of iron. After triggering lobe opening by changing the charge states of tyrosines, the conformations adopted and the iron binding affinity still depend on pH, IS and allosteric interactions. To relate the observed conformational changes to the environmental conditions that might be encountered in the periplasmic space, we offer a plausible model that couples electrostatic potential distribution to the mechanical motions invoked. Although low pH/IS and allosteric perturbations decrease the affinity of iron, it remains high for spontaneous dissociation. However, the conformational changes modulated by the environmental conditions expose iron for chelation. Our study provides a quantitative dimension and molecular details to interpret the contribution of possible environmental conditions present in the periplasmic space to iron dissociation from FbpA, opening up the opportunity of modulating function via allosteric mutations or altering environmental conditions, thus offering a new route to developing strategies towards antibiotic resistance by targeting nutritional requirements.