Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

Genotype-phenotype correlations and disease mechanisms in PEX13-related Zellweger spectrum disorders.

BACKGROUND: Pathogenic variants in PEX-genes can affect peroxisome assembly and function and cause Zellweger spectrum disorders (ZSDs), characterized by variable phenotypes in terms of disease severity, age of onset and clinical presentations. So far, defects in at least 15 PEX-genes have been implicated in Mendelian diseases, but in some of the ultra-rare ZSD subtypes genotype-phenotype correlations and disease mechanisms remain elusive. METHODS: We report five families carrying biallelic variants in PEX13. The identified variants were initially evaluated by using a combination of computational approaches. Immunofluorescence and complementation studies on patient-derived fibroblasts were performed in two patients to investigate the cellular impact of the identified mutations. RESULTS: Three out of five families carried a recurrent p.Arg294Trp non-synonymous variant. Individuals affected with PEX13-related ZSD presented heterogeneous clinical features, including hypotonia, developmental regression, hearing/vision impairment, progressive spasticity and brain leukodystrophy. Computational predictions highlighted the involvement of the Arg294 residue in PEX13 homodimerization, and the analysis of blind docking predicted that the p.Arg294Trp variant alters the formation of dimers, impairing the stability of the PEX13/PEX14 translocation module. Studies on muscle tissues and patient-derived fibroblasts revealed biochemical alterations of mitochondrial function and identified mislocalized mitochondria and a reduced number of peroxisomes with abnormal PEX13 concentration. CONCLUSIONS: This study expands the phenotypic and mutational spectrum of PEX13-related ZSDs and also highlight a variety of disease mechanisms contributing to PEX13-related clinical phenotypes, including the emerging contribution of secondary mitochondrial dysfunction to the pathophysiology of ZSDs.

Accurate Estimation of the Entropy of Rotation-Translation Probability Distributions.

The estimation of rotational and translational entropies in the context of ligand binding has been the subject of long-time investigations. The high dimensionality (six) of the problem and the limited amount of sampling often prevent the required resolution to provide accurate estimates by the histogram method. Recently, the nearest-neighbor distance method has been applied to the problem, but the solutions provided either address rotation and translation separately, therefore lacking correlations, or use a heuristic approach. Here we address rotational-translational entropy estimation in the context of nearest-neighbor-based entropy estimation, solve the problem numerically, and provide an exact and an approximate method to estimate the full rotational-translational entropy.

Conformational Changes of Trialanine in Water Induced by Vibrational Relaxation of the Amide I Mode.

Most of the protein-based diseases are caused by anomalies in the functionality and stability of these molecules. Experimental and theoretical studies of the conformational dynamics of proteins are becoming in this respect essential to understand the origin of these anomalies. However, a description of the conformational dynamics of proteins based on mechano-energetic principles still remains elusive because of the intrinsic high flexibility of the peptide chains, the participation of weak noncovalent interactions, and the role of the ubiquitous water solvent. In this work, the conformational dynamics of trialanine dissolved in water (D2O) is investigated through Molecular Dynamics (MD) simulations combined with instantaneous normal modes (INMs) analysis both at equilibrium and after the vibrational excitation of the C-terminal amide I mode. The conformational equilibrium between α and pPII conformers is found to be altered by the intramolecular relaxation of the amide I mode as a consequence of the different relaxation pathways of each conformer which modify the amount of vibrational energy stored in the torsional motions of the tripeptide, so the α → pPII and pPII → α conversion rates are increased differently. The selectivity of the process comes from the shifts of the vibrational frequencies with the conformational changes that modify the resonance conditions driving the intramolecular energy flows.

Distance-Based Configurational Entropy of Proteins from Molecular Dynamics Simulations.

Estimation of configurational entropy from molecular dynamics trajectories is a difficult task which is often performed using quasi-harmonic or histogram analysis. An entirely different approach, proposed recently, estimates local density distribution around each conformational sample by measuring the distance from its nearest neighbors. In this work we show this theoretically well grounded the method can be easily applied to estimate the entropy from conformational sampling. We consider a set of systems that are representative of important biomolecular processes. In particular: reference entropies for amino acids in unfolded proteins are obtained from a database of residues not participating in secondary structure elements;the conformational entropy of folding of ß2-microglobulin is computed from molecular dynamics simulations using reference entropies for the unfolded state;backbone conformational entropy is computed from molecular dynamics simulations of four different states of the EPAC protein and compared with order parameters (often used as a measure of entropy);the conformational and rototranslational entropy of binding is computed from simulations of 20 tripeptides bound to the peptide binding protein OppA and of ß2-microglobulin bound to a citrate coated gold surface. This work shows the potential of the method in the most representative biological processes involving proteins, and provides a valuable alternative, principally in the shown cases, where other approaches are problematic.

A method for analyzing the vibrational energy flow in biomolecules in solution.

A method is proposed to analyze the intra- and intermolecular vibrational energy flow occurring in biomolecules in solution during relaxation processes. It is based on the assumption that the total energy exchanged between the vibrational modes is minimal and the global process is essentially statistical. This statistical minimum flow method is shown to provide very useful information about the amount and the rate at which energy is transferred between the individual vibrations of the molecule. To demonstrate the performance of the method, an application is made to the relaxation of the amide I mode of N-methylacetamide-d in aqueous D(2)O solution which yields a detailed quantitative description of the process.

Instantaneous normal modes, resonances, and decay channels in the vibrational relaxation of the amide I mode of N-methylacetamide-D in liquid deuterated water.

A nonequilibrium molecular dynamics (MD) study of the vibrational relaxation of the amide I mode of deuterated N-methylacetamide (NMAD) in aqueous (D(2)O) solution is carried out using instantaneous normal modes (INMs). The identification of the INMs as they evolve over time, which is necessary to analyze the energy fluxes, is made by using a novel algorithm which allows us to assign unequivocally each INM to an individual equilibrium normal mode (ENM) or to a group of ENMs during the MD simulations. The time evolution of the energy stored in each INM is monitored and the occurrence of resonances during the relaxation process is then investigated. The decay of the amide I mode, initially excited with one vibrational quantum, is confirmed to fit well to a biexponential function, implying that the relaxation process involves at least two mechanisms with different rate constants. By freezing the internal motions of the solvent, it is shown that the intermolecular vibration-vibration channel to the bending modes of the solvent is closed. The INM analysis reveals then the existence of a major and faster decay channel, which corresponds to an intramolecular vibrational redistribution process and a minor, and slower, decay channel which involves the participation of the librational motions of the solvent. The faster relaxation pathway can be rationalized in turn using a sequential kinetic mechanism of the type P-->M+L-->L, where P (parent) is the initially excited amide I mode, and M (medium) and L (low) are specific midrange and lower-frequency NMAD vibrational modes, respectively.