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.

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.

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.

Coupling between protonation and conformation in cytochrome c oxidase: Insights from constant-pH MD simulations.

Cytochrome c oxidases (CcOs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes reduce dioxygen (O(2)) to water and, simultaneously, generate a transmembrane electrochemical proton gradient. Despite their importance in the aerobic metabolism and the large amount of structural and biochemical data available for the A1-type CcO family, there is still no consensually accepted description of the molecular mechanisms operating in this protein. A substantial number of questions about the CcO's working mechanism remain to be answered, including how the protonation behavior of some key residues is modulated during a reduction cycle and how is the conformation of the protein affected by protonation. The main objective of this work was to study the protonation-conformation coupling in CcOs and identify the molecular factors that control the protonation state of some key residues. In order to directly capture the interplay between protonation and conformational effects, we have performed constant-pH MD simulations of an A1-type CcO inserted into a lipid bilayer in two redox states (oxidized and reduced) at physiological pH. From the simulations, we were able to identify several groups with unusual titration behavior that are highly dependent on the protein redox state, including the A-propionate from heme a and the D-propionate from heme a3, two key groups possibly involved in proton pumping. The protonation state of these two groups is heavily influenced by subtle conformational changes in the protein (notably of R481(I) and R482(I)) and by small changes in the hydrogen bond network.

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.

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism.

Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.

Constant-pH Molecular Dynamics Study of Kyotorphin in an Explicit Bilayer.

To our knowledge, we present the first constant-pH molecular dynamics study of the neuropeptide kyotorphin in the presence of an explicit lipid bilayer. The overall conformation freedom of the peptide was found to be affected by the interaction with the membrane, in accordance with previous results using different methodologies. Analysis of the interactions between the N-terminus amine group of the peptide and several lipid atoms shows that the membrane is able to stabilize both ionized and neutral forms of kyotorphin, resulting in a pKa value that is similar to the one obtained in water. This illustrates how a detailed molecular model of the membrane leads to rather different results than would be expected from simply regarding it as a low-dielectric slab.

Exploring O2 diffusion in A-type cytochrome c oxidases: molecular dynamics simulations uncover two alternative channels towards the binuclear site.

Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

The Effect of Membrane Environment on Surfactant Protein C Stability Studied by Constant-pH Molecular Dynamics.

Pulmonary surfactant protein C (SP-C) is a small peptide with two covalently linked fatty acyl chains that plays a crucial role in the formation and stabilization of the pulmonary surfactant reservoirs during the compression and expansion steps of the respiratory cycle. Although its function is known to be tightly related to its highly hydrophobic character and key interactions maintained with specific lipid components, much is left to understand about its molecular mechanism of action. Also, although it adopts a mainly helical structure while associated with the membrane, factors as pH variation and deacylation have been shown to affect its stability and function. In this work, the conformational behavior of both the acylated and deacylated SP-C isoforms was studied in a DPPC bilayer under different pH conditions using constant-pH molecular dynamics simulations. Our findings show that both protein isoforms are remarkably stable over the studied pH range, even though the acylated isoform exhibits a labile helix-turn-helix motif rarely observed in the other isoform. We estimate similar tilt angles for the two isoforms over the studied pH range, with a generally higher degree of internalization of the basic N-terminal residues in the deacylated case, and observe and discuss some protonation-conformation coupling effects. Both isoforms establish contacts with the surrounding lipid molecules (preferentially with the sn-2 ester bonds) and have a local effect on the conformational behavior of the surrounding lipid molecules, the latter being more pronounced for acylated SP-C.

Changes in spatial extent and peak double optical density of human macular pigment with age.

The purpose of the present work was to estimate the changes in spatial distribution and optical density of macular pigment (MP) with age. A fundus imaging system with high spatial and spectral resolution was adapted to form an indirect ophthalmoscope. The double optical density at 490 nm of the MP as a function of the location in the retina was obtained for 33 healthy subjects (ages: 21-60 years). There was an increase in spatial extent and decrease in double optical density with age. Furthermore, the spatial distribution of MP showed central areas with irregular shapes and a tendency toward asymmetry.

Structural effects of pH and deacylation on surfactant protein C in an organic solvent mixture: a constant-pH MD study.

The pulmonary surfactant protein C (SP-C) is a small highly hydrophobic protein that adopts a mainly helical structure while associated with the membrane but misfolds into a ß-rich metastable structure upon deacylation, membrane dissociation, and exposure to the neutral pH of the aqueous alveolar subphase, eventually leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. The present constant-pH MD study of the acylated and deacylated isoforms of SP-C in a chloroform/methanol/water mixture, often used to mimic the membrane environment, shows that the loss of the acyl groups has a structural destabilizing effect and that the increase of pH promotes intraprotein contacts which contribute to the loss of helical structure in solution. These contacts result from the poor solvation of charged groups by the solvent mixture, which exhibits a limited membrane-mimetic character. Although a single SP-C molecule was used in the simulations, we propose that analogous intermolecular interactions may play a role in the early stages of the protein misfolding and aggregation in this mixture.

A molecular perspective on nonaqueous biocatalysis: contributions from simulation studies.

The technological value of nonaqueous enzymology has been recognized for more than thirty years. A detailed understanding of the molecular determinants of enzyme behaviour in nonaqueous media is essential to explore their potential. Computer simulations have provided valuable contributions to this field, having elucidated how the solvent affects the structural and dynamic properties of enzymes, as well as their activity and enantioselectivity. They have also helped to shed light on the effect of hydration and the role of counterions. In this perspective, we describe the major challenges and achievements of molecular simulations of enzymes in different types of nonaqueous solvents, including organic solvents, ionic liquids and supercritical fluids.

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.

Inter-domain communication mechanisms in an ABC importer: a molecular dynamics study of the MalFGK2E complex.

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase domains is "transmitted" to the transmembrane domains. In this work, we focus our attention on the consequences of hydrolysis and inorganic phosphate exit in the maltose uptake system (MalFGK(2)E) from Escherichia coli. The prime goal is to identify and map the structural changes occurring during an ATP-hydrolytic cycle. For that, we use extensive molecular dynamics simulations to study three potential intermediate states (with 10 replicates each): an ATP-bound, an ADP plus inorganic phosphate-bound and an ADP-bound state. Our results show that the residues presenting major rearrangements are located in the A-loop, in the helical sub-domain, and in the "EAA motif" (especially in the "coupling helices" region). Additionally, in one of the simulations with ADP we were able to observe the opening of the NBD dimer accompanied by the dissociation of ADP from the ABC signature motif, but not from its corresponding P-loop motif. This work, together with several other MD studies, suggests a common communication mechanism both for importers and exporters, in which ATP-hydrolysis induces conformational changes in the helical sub-domain region, in turn transferred to the transmembrane domains via the "coupling helices".

Water encapsulation in a polyoxapolyaza macrobicyclic compound.

A new heteroditopic macrobicyclic compound (t(2)pN(5)O(3)) containing two separate polyoxa and polyaza compartments was synthesized in good yield through a [1 + 1] "tripod-tripod coupling" strategy. The X-ray crystal structure of H(3)t(2)pN(5)O(3)(3+) revealed the presence of one encapsulated water molecule accepting two hydrogen bonds from two protonated secondary amines and donating a hydrogen bond to one amino group. The acid-base behavior of the compound was studied by potentiometry at 298.2 K in aqueous solution and at ionic strength 0.10 M in KCl. The results revealed unusual protonation behavior, namely a surprisingly low fourth protonation constant contrary to what was expected for the compound. (1)H NMR and DOSY experiments, as well as molecular modeling studies, showed that the water encapsulation and the conformation observed in the solid state are retained in solution. The strong binding of the encapsulated water molecule, reinforced by the cooperative occurrence of a trifurcated hydrogen bond at the polyether compartment of the macrobicycle, account for the very low log K(4)(H) value obtained.

Analyzing the molecular basis of enzyme stability in ethanol/water mixtures using molecular dynamics simulations.

One of the drawbacks of nonaqueous enzymology is the fact that enzymes tend to be less stable in organic solvents than in water. There are, however, some enzymes that display very high stabilities in nonaqueous media. In order to take full advantage of the use of nonaqueous solvents in enzyme catalysis, it is essential to elucidate the molecular basis of enzyme stability in these media. Toward this end, we performed µs-long molecular dynamics simulations using two homologous proteases, pseudolysin, and thermolysin, which are known to have considerably different stabilities in solutions containing ethanol. The analysis of the simulations indicates that pseudolysin is more stable than thermolysin in ethanol/water mixtures and that the disulfide bridge between C30 and C58 is important for the stability of the former enzyme, which is consistent with previous experimental observations. Our results indicate that thermolysin has a higher tendency to interact with ethanol molecules (especially through van der Waals contacts) than pseudolysin, which can lead to the disruption of intraprotein hydrophobic interactions and ultimately result in protein unfolding. In the absence of the C30-C58 disulfide bridge, pseudolysin undergoes larger conformational changes, becoming more open and more permeable to ethanol molecules which accumulate in its interior and form hydrophobic interactions with the enzyme, destroying its structure. Our observations are not only in good agreement with several previous experimental findings on the stability of the enzymes studied in ethanol/water mixtures but also give an insight on the molecular determinants of this stability. Our findings may, therefore, be useful in the rational development of enzymes with increased stability in these media.

Structural determinants for the membrane insertion of the transmembrane peptide of hemagglutinin from influenza virus.

Membrane fusion is a process involved in a high range of biological functions, going from viral infections to neurotransmitter release. Fusogenic proteins increase the slow rate of fusion by coupling energetically downhill conformational changes of the protein to the kinetically unfavorable fusion of the membrane lipid bilayers. Hemagglutinin is an example of a fusogenic protein, which promotes the fusion of the membrane of the influenza virus with the membrane of the target cell. The N-terminus of the HA2 subunit of this protein contains a fusion domain described to act as a destabilizer of the target membrane bilayers, leading eventually to a full fusion of the two membranes. On the other hand, the C-terminus of the same subunit contains a helical transmembrane domain which was initially described to act as the anchor of the protein to the membrane of the virus. However, in recent years the study of this peptide segment has been gaining more attention since it has also been described to be involved in the membrane fusion process. Yet, the structural characterization of the interaction of such a protein domain with membrane lipids is still very limited. Therefore, in this work, we present a study of this transmembrane peptide domain in the presence of DMPC membrane bilayers, and we evaluate the effect of several mutations, and the effect of peptide oligomerization in this interaction process. Our results allowed us to identify and confirm amino acid residue motifs that seem to regulate the interaction between the segment peptide and membrane bilayers. Besides these sequence requirements, we have also identified length and tilt requirements that ultimately contribute to the hydrophobic matching between the peptide and the membrane. Additionally, we looked at the association of several transmembrane peptide segments and evaluated their direct interaction and stability inside a membrane bilayer. From our results we could conclude that three independent TM peptide segments arrange themselves in a parallel arrangement, very similarly to what is observed for the C-terminal regions of the hemagglutinin crystallographic structure of the protein, to where the segments are attached.

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.

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.

Is the prediction of pKa values by constant-pH molecular dynamics being hindered by inherited problems?

In this study, we investigate two factors that can hinder the performance of constant-pH molecular dynamics methods in predicting protein pK(a) values, using hen egg white lysozyme as a test system. The first factor is related to the molecular definition and pK(a) value of model compounds in the Poisson-Boltzmann framework. We address this by defining the model compound as a molecular fragment with an associated pK(a) value that is calibrated against experimental data, which results in a decrease of 0.12 units in pK(a) errors. The second addressed factor is the possibility that detrimental structural distortions are being introduced in the simulations by the underlying molecular mechanics force field. This issue is investigated by analyzing how the gradual structural rearrangements affect the predicted pK(a) values. The two GROMOS force fields studied here (43A1 and 53A6) yield good pK(a) predictions, although a time-dependent performance is observed: 43A1 performs better after a few nanoseconds of structural reorganization (pK(a) errors of ~0.45), while 53A6 gives the best prediction right at the first nanosecond (pK(a) errors of 0.42). These results suggest that the good performance of constant-pH molecular dynamics methods could be further improved if these force field limitations were overcome.