The effect of cycloplegia in the accuracy of autorefraction, keratometry and axial length using the Myopia Master.

BACKGROUND: Assessing refractive errors under cycloplegia is recommended for paediatric patients; however, this may not always be feasible. In these situations, refraction has to rely on measurements made under active accommodation which may increase measurements variability and error. Therefore, evaluating the accuracy and precision of non-cycloplegic refraction and biometric measurements is clinically relevant. The Myopia Master, a novel instrument combining autorefraction and biometry, is designed for monitoring refractive error and ocular biometry in myopia management. This study assessed its repeatability and agreement for autorefraction and biometric measurements pre- and post-cycloplegia. METHODS: A prospective cross-sectional study evaluated a cohort of 96 paediatric patients that underwent ophthalmologic examination. An optometrist performed two repeated measurements of autorefraction and biometry pre- and post-cycloplegia. Test-retest repeatability (TRT) was assessed as differences between consecutive measurements and agreement as differences between post- and pre-cycloplegia measurements, for spherical equivalent (SE), refractive and keratometric J0/J45 astigmatic components, mean keratometry (Km) and axial length (AL). RESULTS: Cycloplegia significantly improved the SE repeatability (TRT, pre-cyclo: 0.65 D, post-cyclo: 0.31 D). SE measurements were more repeatable in myopes and emmetropes compared to hyperopes. Keratometry (Km) repeatability did not change with cycloplegia (TRT, pre-cyclo: 0.25 D, post-cyclo:0.27 D) and AL repeatability improved marginally (TRT, pre-cyclo: 0.14 mm, post-cyclo: 0.09 mm). Regarding pre- and post-cycloplegia agreement, SE became more positive by + 0.79 D, varying with refractive error. Myopic eyes showed a mean difference of + 0.31 D, while hyperopes differed by + 1.57 D. Mean keratometry, refractive and keratometric J0/J45 and AL showed no clinically significant differences. CONCLUSIONS: Refractive error measurements, using the Myopia Master were 2.5x less precise pre-cycloplegia than post-cycloplegia. Accuracy of pre-cycloplegic refractive error measurements was often larger than the clinically significant threshold (0.25 D) and was refractive error dependent. The higher precision compared to autorefraction measurements, pre- and post-cycloplegia agreement and refractive error independence of AL measurements emphasize the superiority of AL in refractive error monitoring.

Repeatability and agreement of swept-source optical coherence tomography and partial coherence interferometry biometers in myopes.

CLINICAL RELEVANCE: Biometric measurements in the context of myopia are fundamental to detect eyes at risk of developing myopia and during the follow-up of patients with myopia control treatment. Thus, the accuracy of biometers has high clinical relevance. BACKGROUND: The Myopia Master is a new biometer based on partial coherence interferometry especially dedicated to the follow-up of myopic patients. This study aims to assess the repeatability of the Myopia Master and evaluate its agreement with a swept-source optical coherence interferometry biometer (IOL Master 700). METHODS: This cross-sectional prospective study assessed the biometric parameters of two groups of myopes (age range: 8-16 years old), spectacle corrected (n = 60) and orthokeratology contact lens wearers (n = 60). One senior optometrist performed two consecutive measurements per instrument, which included axial length, mean keratometry and horizontal visible iris diameter (HVID). The repeatability of each device and the agreement between devices were assessed by the dispersion of the measurement differences, for AL, mean keratometry, corneal astigmatism and HVID. RESULTS: The two biometers measured approximately the same value in both measurements. Test-retest repeatability tended to be lower than clinical significant thresholds, in particular, for AL and mean keratometry. Corneal-related parameters tended to have lower repeatability in the orthokeratology group, especially mean keratometry. The agreement between instruments revealed statistically significant differences between devices with the SS-OCT measuring longer eyes, steeper corneas and larger HVID. CONCLUSIONS: In a paediatric population, the Myopia Master showed clinically acceptable repeatability levels, but the IOL Master 700 demonstrated superior repeatability. Eyes treated with orthokeratology may compromise the repeatability of the corneal-related parameters. The Myopia Master and the IOL Master 700 are repeatable devices appropriate for monitoring myopia progression, but the differences observed do not allow their use interchangeably.

Computational Study of the pH-Dependent Ionic Environment around ß-Lactoglobulin.

Ions are involved in multiple biological processes and may exist bound to biomolecules or may be associated with their surface. Although the presence of ions in nucleic acids has traditionally gained more interest, ion-protein interactions, often with a marked dependency on pH, are beginning to gather attention. Here we present a detailed analysis on the binding and distribution of ions around ß-lactoglobulin using a constant-pH MD (CpHMD) method, at a pH range 3-8, and compare it with the more traditional Poisson-Boltzmann (PB) model and the existing experimental data. Most analyses used ion concentration maps built around the protein, obtained from either the CpHMD simulations or PB calculations. The requirements of approximate charge neutrality and ionic strength equal to bulk, imposed on the MD box, imply that the absolute value of the ion excess should be half the protein charge, which is in agreement with experimental observation on other proteins ( Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2015879118) and lends support to this protocol. In addition, the protein total charge (including territorially bound ions) estimated with MD is in excellent agreement with electrophoretic measurements. Overall, the CpHMD simulations show good agreement with the nonlinear form of the PB (NLPB) model but not with its linear form, which involves a theoretical inconsistency in the calculation of the concentration maps. In several analyses, the observed pH-dependent trends for the counterions and co-ions are those generally expected, and the ion concentration maps correctly converge to the bulk ionic strength as one moves away from the protein. Despite the overall similarity, the CpHMD and NLPB approaches show some discrepancies when analyzed in more detail, which may be related to an apparent overestimation of counterion excess and underestimation of co-ion exclusion by the NLPB model, particularly at short distances from the protein.

FixBox: A General Algorithm to Fix Molecular Systems in Periodic Boxes.

Periodic boundary conditions (PBCs) are a standard feature of molecular simulations, and their mathematical and computational aspects are well-understood and relatively straightforward. However, they can in practice be a nuisance when simulating heterogeneous systems, especially when different types of molecules change their relative positions during the simulation. Although the translation required to fix a broken molecular complex of interest can in most cases be easily inferred by visual inspection, it typically depends on the type of system, its configuration, and the box geometry, making automated procedures problematic. We present here a general algorithm, named FixBox, that can fix a molecular complex of interest from a minimal set of definitions of its assembling parts and intended arrangement in the simulation box. It uses a unified triclinic framework for the box geometric periodicity, does not require a full molecular topology, and is applicable to various types of systems and configurations, making it possible to fully and easily automate the fixing of a broken molecular complex. The performance of the algorithm is illustrated with problematic configurations of various types of simulated systems. The presented formal framework can generally be useful for algorithms that need to perform geometrical transformations on systems with PBCs.

Approach to Study pH-Dependent Protein Association Using Constant-pH Molecular Dynamics: Application to the Dimerization of ß-Lactoglobulin.

Protein-protein association is often mediated by electrostatic interactions and modulated by pH. However, experimental and computational studies have often overlooked the effect of association on the protonation state of the protein. In this work, we present a methodological approach based on constant-pH molecular dynamics (MD), which aims to provide a detailed description of a pH-dependent protein-protein association, and apply it to the dimerization of ß-lactoglobulin (BLG). A selection of analyses is performed using the data generated by constant-pH MD simulations of monomeric and dimeric forms of bovine BLG, in the pH range 3-8. First, we estimate free energies of dimerization using a computationally inexpensive approach based on the Wyman-Tanford linkage theory, calculated in a new way through the use of thermodynamically based splines. The individual free energy contribution of each titratable site is also calculated, allowing for identification of relevant residues. Second, the correlations between the proton occupancies of pairs of sites are calculated (using the Pearson coefficient), and extensive networks of correlated sites are observed at acidic pH values, sometimes involving distant pairs. In general, strongly correlated sites are also slow proton exchangers and contribute significantly to the pH-dependency of the dimerization free energy. Third, we use ionic density as a fingerprint of protein charge distribution and observe electrostatic complementarity between the monomer faces that form the dimer interface, more markedly at the isoionic point (where maximum dimerization occurs) than at other pH values, which might contribute to guide the association. Finally, the pH-dependent dimerization modes are inspected using PCA, among other analyses, and two states are identified: a relaxed state at pH 4-8 (with the typical alignment of the crystallographic structure) and a compact state at pH 3-4 (with a tighter association and rotated alignment). This work shows that an approach based on constant-pH MD simulations can produce rich detailed pictures of pH-dependent protein associations, as illustrated for BLG dimerization.

Association between Clinical Vision Measures and Visual Perception and Soccer Referees' On-field Performance.

SIGNIFICANCE: The decisions taken by soccer officials are critically important to game management. Understanding the underlying processes that mediate expert performance in soccer refereeing may lead to a better standard of officiating. Vision is the dominant source of incoming information upon which officials rely to make their on-field decisions. PURPOSE: We tested the hypothesis that performance on generic tests of vision and visual perception predicts domain-specific performance in elite-level soccer referees (R) and assistant referees (AR). METHODS: We assessed the vision of R and AR who officiate at the highest level in Portugal. To be eligible for inclusion, R and AR had to have officiated for at least two consecutive seasons across the 2014/2015, 2015/2016, and 2016/2017 seasons. A single, rank-order list of the performance of eligible officials was created based on the rank-order list for each season that was made by the Portuguese Soccer Federation. Clinical vision measures included visual acuity and stereoacuity, and visual perception measures were gathered using the Test of Visual Perceptual Skills, Third Edition. RESULTS: A total of 59 officials participated (21 R, 38 AR), 17 of whom officiated at the international level. The R and AR groups did not differ in vision or visual perception measures. We found that better stereoacuity (P < .001) and visual memory (P = .001) are associated with a higher rank order of on-field performance after adjusting for the age, experience, the national/international status, and the regional affiliation of the officials. Together, these two measures explain 22% of the variance in rank-order performance. CONCLUSIONS: This is the first study to show a link between the vision of officials and their on-field performance. The origin and significance of these findings remain to be established, and further work is required to establish whether they are component skills in the domain of soccer refereeing.

Effect of pH on the influenza fusion peptide properties unveiled by constant-pH molecular dynamics simulations combined with experiment.

The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide's effect on the membrane.

Inter-Eye Vault Differences of Implantable Collamer Lens Measured Using Anterior Segment Optical Coherence Tomography.

PURPOSE: The distance between an implantable collamer lens (ICL) and the crystalline lens, namely vault, is a space regulated by the interaction of the ICL and the anatomical structures of the eye. This study analysed the differences in vault size between fellow eyes with similar anterior segment biometry. PATIENTS AND METHODS: A retrospective case series analysed 109 cases of patients bilaterally implanted with EVO-V4c. Patients were analysed pre- and postoperatively using anterior segment optical coherence tomography. The range of vault inter-eye differences was defined as the 95% confidence interval of the differences. Bivariate correlation was applied to seek for associations between vault inter-eye differences with biometric and lens parameters (angle-to-angle, anterior chamber depth, crystalline lens rise, central corneal thickness, central keratometry, ICL spherical equivalent, horizontal compression, postoperative pupil diameter and vault). RESULTS: Mean vault inter-eye differences were similar between fellow eyes (26.0 ± 122.5 µm). The 95% confidence interval range of the differences was ±240.1 µm, nearly 50% of the cases presented vault inter-eye differences higher than 100 µm. The vault of the first operated eye explained 81% of the variance in the second eye vault. Vault inter-eye differences were positively correlated with the level of horizontal compression and with vault magnitude. CONCLUSION: Vaults measured in fellow eyes may present considerable differences, which can reach 25% of the common vault range. This reflects some degree of baseline variability in the vault. Clinically, these differences assume special relevance in cases where low or high vaults are expected.

Multilevel cervical arthroplasty-clinical and radiological outcomes.

BACKGROUND: Cervical disc arthroplasty (CDA) is a valid option for single-level cervical disc disease (CDD) as an alternative to fusion. However, the use in 3- and 4-level disc disease is under scrutiny with scarce data published so far. Our aim was to study clinical and radiological outcomes of arthroplasty in patients with multilevel CDD. METHODS: Retrospective analysis of clinical records, pre- and post-operative neutral/dynamic X-rays of patients who underwent CDA in multilevel CDD (2-4 levels). We evaluated sagittal balance parameters (C2-7 and index angle, SVA), global and segmental range of motion (ROM)), neck and arm VAS, Odom's criteria, re-operation rate, adjacent segment disease (ASD), willingness to undertake the same procedure again. Rate of heterotopic ossification (HO) was studied for follow-up >2 years. A subgroup analysis was performed regarding 2-level versus 3- or 4-level arthroplasty. RESULTS: Thirty-two patients were included, 6 males and 26 females, mean age of 46 years (range, 30-63). Seventy-seven cervical disc levels were treated with the same artificial disc. Twenty-one patients were operated on 2 levels, nine in 3 levels, and 2 patients in 4 levels. Post-operatively, there was a decrease in SVA (-2.2±8.36 mm, P=0.098) and an increase in global (3.7±9.6º, P=0.042) and index (1.3±6.1º, P=0.071) ROM. Mean nVAS and aVAS decreased (7.5±1.1 to 2.5±1.5; 6.3±1.9 to 2.2±1.7, P<0.05). Two-level versus 3-4 level patient subgroups showed a lower SVA (-1.3±8.1 mm P=0.47; -3.4±6.3 mm P=0.107), a slight increase in global (1.6±9.4º P=0.44; 7.2±11.7º P=0.07) and index (1.1±4.7º P=0.12; 1.3±8.1º P=0.35) ROM. HO was present in 9.9% (7/71) of disc levels operated, none of them with grade 3 or 4. CONCLUSIONS: Multilevel CDA provides good clinical and radiological outcomes, preserving global and segmental cervical mobility, while having a beneficial effect on sagittal balance. These results hold for 2 to 4 levels, making this technique a valuable option in selected patients with cervical multilevel CDD.

A pH Replica Exchange Scheme in the Stochastic Titration Constant-pH MD Method.

Solution pH is a physicochemical property that has a key role in cellular regulation, and its impact at the molecular level is often difficult to study by experimental methods. In this context, several theoretical methods were developed to study pH effects in macromolecules. The stochastic titration constant-pH molecular dynamics method (CpHMD) was developed by coupling molecular sampling methods, which are appropriate to study the conformational ensemble of biomolecules, with continuum electrostatics approaches, which properly describe pH-dependent protonation states. However, in difficult cases, the protonation sampling can be too slow for the commonly accessible computational times. In this work, we combined a pH replica exchange scheme with this CpHMD method and explored several optimization strategies and possible limitations.

Molecular Anatomy of Plant Photoprotective Switches: The Sensitivity of PsbS to the Environment, Residue by Residue.

Under strong sunlight, plants avoid photooxidation by quenching the excess absorbed energy. Quenching is triggered by PsbS, a membrane protein that is activated and deactivated by the light-dependent pH changes in the thylakoid lumen. The mechanism of action of this protein is unknown, but it was suggested that several glutamates act as pH sensors. However, the p Ka of glutamate is several pH units below the physiological values in the lumen. Thus, how can PsbS sense the pH of the lumen, and how does it respond to it? By applying a nonstandard molecular dynamics method that treats pH explicitly, we show that the lumen-exposed glutamates of PsbS have strongly shifted p Ka values and that such shifts are crucial for the pH sensitivity in physiological conditions. We also demonstrate that protonation drives a systematic unfolding of a region key for protein-protein interactions, indicating that PsbS response to pH is a functional conformational switch.

Role of Counterions in Constant-pH Molecular Dynamics Simulations of PAMAM Dendrimers.

Electrostatic interactions play a pivotal role in the structure and mechanism of action of most biomolecules. There are several conceptually different methods to deal with electrostatics in molecular dynamics simulations. Ionic strength effects are usually introduced using such methodologies and can have a significant impact on the quality of the final conformation space obtained. We have previously shown that full system neutralization can lead to wrong lipidic phases in the 25% PA/PC bilayer (J. Chem. Theory Comput. 2014,10, 5483-5492). In this work, we investigate how two limit approaches to the ionic strength treatment (implicitly with GRF or using full system neutralization with either GRF or PME) can influence the conformational space of the second-generation PAMAM dendrimer. Constant-pH MD simulations were used to map PAMAM's conformational space at its full pH range (from 2.5 to 12.5). Our simulations clearly captured the coupling between protonation and conformation in PAMAM. Interestingly, the dendrimer conformational distribution was almost independent of the ionic strength treatment methods, which is in contrast to what we have observed in charged lipid bilayers. Overall, our results confirm that both GRF with implicit ionic strength and a fully neutralized system with PME are valid approaches to model charged globular systems, using the GROMOS 54A7 force field.

Conservation planning for freshwater-marine carryover effects on Chinook salmon survival.

Experiences of migratory species in one habitat may affect their survival in the next habitat, in what is known as carryover effects. These effects are especially relevant for understanding how freshwater experience affects survival in anadromous fishes. Here, we study the carryover effects of juvenile salmon passage through a hydropower system (Snake and Columbia rivers, northwestern United States). To reduce the direct effect of hydrosystem passage on juveniles, some fishes are transported through the hydrosystem in barges, while the others are allowed to migrate in-river. Although hydrosystem survival of transported fishes is greater than that of their run-of-river counterparts, their relative juvenile-to-adult survival (hereafter survival) can be less. We tested for carryover effects using generalized linear mixed effects models of survival with over 1 million tagged Chinook salmon, Oncorhynchus tshawytscha (Walbaum) (Salmonidae), migrating in 1999-2013. Carryover effects were identified with rear-type (wild vs. hatchery), passage-type (run-of-river vs. transported), and freshwater and marine covariates. Importantly, the Pacific Decadal Oscillation (PDO) index characterizing cool/warm (i.e., productive/nonproductive) ocean phases had a strong influence on the relative survival of rear- and passage-types. Specifically, transportation benefited wild Chinook salmon more in cool PDO years, while hatchery counterparts benefited more in warm PDO years. Transportation was detrimental for wild Chinook salmon migrating early in the season, but beneficial for later season migrants. Hatchery counterparts benefited from transportation throughout the season. Altogether, wild fish could benefit from transportation approximately 2 weeks earlier during cool PDO years, with still a benefit to hatchery counterparts. Furthermore, we found some support for hypotheses related to higher survival with increased river flow, high predation in the estuary and plume areas, and faster migration and development-related increased survival with temperature. Thus, pre- and within-season information on local- and broad-scale conditions across habitats can be useful for planning and implementing real-time conservation programs.

Factors affecting the bacterial community composition and heterotrophic production of Columbia River estuarine turbidity maxima.

Estuarine turbidity maxima (ETM) function as hotspots of microbial activity and diversity in estuaries, yet, little is known about the temporal and spatial variability in ETM bacterial community composition. To determine which environmental factors affect ETM bacterial populations in the Columbia River estuary, we analyzed ETM bacterial community composition (Sanger sequencing and amplicon pyrosequencing of 16S rRNA gene) and bulk heterotrophic production (3 H-leucine incorporation rates). We collected water 20 times to cover five ETM events and obtained 42 samples characterized by different salinities, turbidities, seasons, coastal regimes (upwelling vs. downwelling), locations, and particle size. Spring and summer populations were distinct. All May samples had similar bacterial community composition despite having different salinities (1-24 PSU), but summer non-ETM bacteria separated into marine, freshwater, and brackish assemblages. Summer ETM bacterial communities varied depending on coastal upwelling or downwelling conditions and on the sampling site location with respect to tidal intrusion during the previous neap tide. In contrast to ETM, whole (>0.2 µm) and free-living (0.2-3 µm) assemblages of non-ETM waters were similar to each other, indicating that particle-attached (>3 µm) non-ETM bacteria do not develop a distinct community. Brackish water type (ETM or non-ETM) is thus a major factor affecting particle-attached bacterial communities. Heterotrophic production was higher in particle-attached than free-living fractions in all brackish waters collected throughout the water column during the rise to decline of turbidity through an ETM event (i.e., ETM-impacted waters). However, free-living communities showed higher productivity prior to or after an ETM event (i.e., non-ETM-impacted waters). This study has thus found that Columbia River ETM bacterial communities vary based on seasons, salinity, sampling location, and particle size, with the existence of three particle types characterized by different bacterial communities in ETM, ETM-impacted, and non-ETM-impacted brackish waters. Taxonomic analysis suggests that ETM key biological function is to remineralize organic matter.

Visual and health outcomes, measured with the activity inventory and the EQ-5D, in visual impairment.

PURPOSE: Generic instruments to assess health utilities can be used to express the burden of health problems in widely used indexes. That is in contrast with what can be obtained with condition-specific instruments, outcomes are very specific and difficult to compare across conditions. The purpose of this study was to assess health and visual outcomes and its determinants in patients with visual impairment (VI) using the EQ-5D-3L and the Activity Inventory (AI). METHODS: Participants were recruited in different hospitals during the PCVIP-study. A total of 134 patients with acuity 0.30 logMAR or less in the better eye were interviewed. The AI includes 46 goals split between three objectives: social functioning, recreation and daily living, and was used to measure visual ability. The EQ-5D consists of five questions covering one domain each and was used to provide a measure of health states. Responses to each domain were combined to produce a single individual index. RESULTS: The AI and the EQ-5D-3L showed enough discriminatory power between VI levels (p < 0.001), and their results were strongly correlated r(134) = 0.825, (p < 0.001). Explanatory factors for visual ability were level of VI in better eye, age and gender, R2  = 0.43, (p < 0.001). Explanatory factors for the EQ-5D-3L were level of VI in the better eye, comorbidities and gender, R2  = 0.36, (p < 0.001). CONCLUSION: Our results showed that the EQ-5D-3L is useful when characterizing the burden of VI and to compute, when necessary, quality-adjusted-life-years (QALY) changes due to VI. However, it is important to consider that the EQ-5D-3L uses a coarse response scale, assesses a limited spectrum of domains and is influenced by comorbidities. This might limit its responsiveness to small changes in visual ability.

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study.

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

Predicting protein partition coefficients in aqueous two phase system.

The present work aims to achieve an additional insight into the protein partitioning behavior in aqueous two phase systems (ATPSs), together with a study on the viability of a semi-empirical model based on continuum electrostatics to predict the protein partition characteristics. The partitioning behaviors of 14 globular proteins, with different properties, were explored in three polymer/polymer ATPSs. By the Collander equation, a linear correlation between protein partitioning coefficients in all systems was observed. Using the semi-empirical model it was possible to predict the partitioning behavior of proteins. The electrostatic energy depends on the protein size and ATPSs characteristics and varies in agreement with the difference in phase dielectric constants. Linear correlation of nonpolar energy, and the solvent accessible surface area was observed. Polymer structure and concentration have a significant influence on model viability. A good qualitative prediction of preferred phase for studied proteins was observed.

Exploring the Structural Properties of Positively Charged Peptide Dendrimers.

We report a combined experimental and computational approach to study the structural behavior of positively charged peptide dendrimers. Third-generation dendrimers containing combinations of positive/neutral amino acid residues in the different dendrimer generations were synthesized and their overall size evaluated using diffusion NMR. Molecular dynamics simulations were performed to obtain a comprehensive description of the molecular-level phenomena substantiating the structural differences observed. Comparison of the results presented with previous findings reveals a striking charge-dependent tendency in these systems, where the simple number and placement of charged amino acids in the sequence allows an extensive control over the exhibited structural features. Indeed, we observe that peptide dendrimers bearing progressively higher amounts of charged residues are characterized by an increasing structural plasticity, with a myriad of conformational states equally accessible to them. On the other hand, dendrimers containing only small amounts of charged residues evidence, to some extent, a characteristic structural rigidity.

Structuring Peptide Dendrimers through pH Modulation and Substrate Binding.

Dendrimers are a family of ramified synthetic molecules. pH effects and electrostatic interactions are known to be crucial players to explain the conformational and functional behaviors observed in these systems. Nonetheless, to date, no computational study involving these systems has explicitly addressed the protonation equilibrium taking place at different pH values for dendrimers containing multiple ionizable sites. Herein, we present the results of constant-pH molecular dynamics simulations performed at several pH values for four peptide dendrimers of different generations (from one to four) composed of the same type of amino acids: histidines, serines, and diaminopropionic acid. These dendrimers are known to catalyze the hydrolysis of pyrene sulfonate esters. Constant-pH MD simulations in the presence of substrate molecules at the optimum pH for catalysis are also reported. The results show that first and second generation dendrimers are almost structurally unresponsive to pH variations. For third and fourth generation dendrimers, pH plays a structuring role, with markedly different behaviors being observed when passing from acidic to neutral pH. Protonation-conformation coupling effects influence several intramolecular interactions, which, in turn, modulate the shape and structure at the different pH values. The atypical and highly pH-dependent protonation profiles of some histidine residues are also investigated. The interactions between dendrimers and substrates restrict the conformational space available to the dendrimers and enforce conformational homogeneity. This structuring effect is a consequence of the dendrimer-substrate interactions which occur through stabilizing hydrogen bonds and ion pairs between the substrate sulfonate groups and the dendrimer residues. Our results provide original fundamental data contributing to the development of novel pH-modulated dendritic systems and the improvement of the existing ones.