Estimation of protein folding probability from equilibrium simulations

The assumption that similar structures have similar folding probabilities ($p_{fold}$) leads naturally to a procedure to evaluate $p_{fold}$ for every snapshot saved along an equilibrium folding-unfolding trajectory of a structured peptide or protein. The procedure utilizes a structurally homogeneous clustering and does not require any additional simulation. It can be used to detect multiple folding pathways as shown for a three-stranded antiparallel $\beta$-sheet peptide investigated by implicit solvent molecular dynamics simulations.Comment: 7 pages, 4 figures, supplemetary material

Synergistic Allostery in Multiligand-Protein Interactions

Amide hydrogen-deuterium exchange mass spectrometry is powerful for describing combinatorial coupling effects of a cooperative ligand pair binding at noncontiguous sites: adenosine at the ATP-pocket and a docking peptide (PIFtide) at the PIF-pocket, on a model protein kinase PDK1. Binding of two ligands to PDK1 reveal multiple hotspots of synergistic allostery with cumulative effects greater than the sum of individual effects mediated by each ligand. We quantified this synergism and ranked these hotspots using a difference in deuteration-based approach, which showed that the strongest synergistic effects were observed at three of the critical catalytic loci of kinases: the αB-αC helices, and HRD-motif loop, and DFG-motif. Additionally, we observed weaker synergistic effects at a distal GHI-subdomain locus. Synergistic changes in deuterium exchange observed at a distal site but not at the intermediate sites of the large lobe of the kinase reveals allosteric propagation in proteins to operate through two modes. Direct electrostatic interactions between polar and charged amino acids that mediate targeted relay of allosteric signals, and diffused relay of allosteric signals through soft matter-like hydrophobic core amino acids. Furthermore, we provide evidence that the conserved β-3 strand lysine of protein kinases (Lys111 of PDK1) functions as an integrator node to coordinate allosteric coupling of the two ligand-binding sites. It maintains indirect interactions with the ATP-pocket and mediates a critical salt bridge with a glutamate (Glu130) of αC helix, which is conserved across all kinases. In summary, allosteric propagation in cooperative, dual-liganded enzyme targets is bidirectional and synergistic and offers a strategy for combinatorial drug development.Fil: Ghode, Abhijeet. National University Of Singapore; SingapurFil: Gross, Lissy Zoe Florens. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Tee, Wei Ven. National University Of Singapore; SingapurFil: Guarnera, Enrico. Agency For Science, Technology And Research; SingapurFil: Berezovsky, Igor N.. Agency For Science, Technology And Research; SingapurFil: Biondi, Ricardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Anand, Ganesh S.. National University Of Singapore; Singapu

AlloMAPS: allosteric mutation analysis and polymorphism of signaling database

AlloMAPS database provides data on the causality and energetics of allosteric communication obtained with the structure-based statistical mechanical model of allostery (SBSMMA). The database contains data on allosteric signaling in three sets of proteins and protein chains: (i) 46 proteins with comprehensively annotated functional and allosteric sites; (ii) 1908 protein chains from PDBselect set of chains with low (<25%) sequence identity; (iii) 33 proteins with more than 50 known pathological SNPs in each molecule. In addition to energetics of allosteric signaling between known functional and regulatory sites, allosteric modulation caused by the binding to these sites, by SNPs, and by mutations designated by the user can be explored. Allosteric Signaling Maps (ASMs), which are produced via the exhaustive computational scanning for stabilizing and destabilizing mutations and for the modulation range caused by the sequence position are available for each protein/protein chain in the database. We propose to use this database for evaluating the effects of allosteric signaling in the search for latent regulatory sites and in the design of allosteric sites and effectors. The database is freely available at: http://allomaps.bii.a-star.edu.sg.Funding for open access charges: Biomedical Research Council, Agency for Science, Technology and Research (A*STAR

AI/ML combined with next-generation sequencing of VHH immune repertoires enables the rapid identification of de novo humanized and sequence-optimized single domain antibodies: a prospective case study

Introduction: In this study, we demonstrate the feasibility of yeast surface display (YSD) and nextgeneration sequencing (NGS) in combination with artificial intelligence and machine learning methods (AI/ML) for the identification of de novo humanized single domain antibodies (sdAbs) with favorable early developability profiles.Methods: The display library was derived from a novel approach, in which VHH-based CDR3 regions obtained from a llama (Lama glama), immunized against NKp46, were grafted onto a humanized VHH backbone library that was diversified in CDR1 and CDR2. Following NGS analysis of sequence pools from two rounds of fluorescence-activated cell sorting we focused on four sequence clusters based on NGS frequency and enrichment analysis as well as in silico developability assessment. For each cluster, long short-term memory (LSTM) based deep generative models were trained and used for the in silico sampling of new sequences. Sequences were subjected to sequence- and structure-based in silico developability assessment to select a set of less than 10 sequences per cluster for production.Results: As demonstrated by binding kinetics and early developability assessment, this procedure represents a general strategy for the rapid and efficient design of potent and automatically humanized sdAb hits from screening selections with favorable early developability profiles

Early Diagnosis, Treatment and Follow-Up of Cystic Echinococcosis in Remote Rural Areas in Patagonia: Impact of Ultrasound Training of Non-Specialists

Cystic echinococcosis (CE) is an important and widespread disease that affects sheep, cattle, and humans living in areas where sheep and cattle are raised. CE is highly endemic in rural sections of Rio Negro, Argentina, where our group is based. However, it requires continuous monitoring of both populations with human disease best assessed by means of ultrasound (US) screening. This is challenging in remote rural areas due to the shortage of imaging specialists. To overcome this hurdle, we set up a two-day training program of Focused Assessment with Sonography for Echinococcosis (FASE) on CE for family medicine practitioners with no previous experience in US. After the course, they were equipped with portable US scanners and dispatched to remote rural areas in Rio Negro where they screened patients, located and staged the cysts and decided on the treatment with the help of surgeons and radiologists in local tertiary care centers

Structure-Based Statistical Mechanical Model Accounts for the Causality and Energetics of Allosteric Communication.

Allostery is one of the pervasive mechanisms through which proteins in living systems carry out enzymatic activity, cell signaling, and metabolism control. Effective modeling of the protein function regulation requires a synthesis of the thermodynamic and structural views of allostery. We present here a structure-based statistical mechanical model of allostery, allowing one to observe causality of communication between regulatory and functional sites, and to estimate per residue free energy changes. Based on the consideration of ligand free and ligand bound systems in the context of a harmonic model, corresponding sets of characteristic normal modes are obtained and used as inputs for an allosteric potential. This potential quantifies the mean work exerted on a residue due to the local motion of its neighbors. Subsequently, in a statistical mechanical framework the entropic contribution to allosteric free energy of a residue is directly calculated from the comparison of conformational ensembles in the ligand free and ligand bound systems. As a result, this method provides a systematic approach for analyzing the energetics of allosteric communication based on a single structure. The feasibility of the approach was tested on a variety of allosteric proteins, heterogeneous in terms of size, topology and degree of oligomerization. The allosteric free energy calculations show the diversity of ways and complexity of scenarios existing in the phenomenology of allosteric causality and communication. The presented model is a step forward in developing the computational techniques aimed at detecting allosteric sites and obtaining the discriminative power between agonistic and antagonistic effectors, which are among the major goals in allosteric drug design

How Does a Simplified-Sequence Protein Fold?

To investigate a putatively primordial protein we have simplified the sequence of a 56-residue α/β fold (the immunoglobulin-binding domain of protein G) by replacing it with polyalanine, polythreonine, and diglycine segments at regions of the sequence that in the folded structure are α-helical, β-strand, and turns, respectively. Remarkably, multiple folding and unfolding events are observed in a 15-μs molecular dynamics simulation at 330 K. The most stable state (populated at ∼20%) of the simplified-sequence variant of protein G has the same α/β topology as the wild-type but shows the characteristics of a molten globule, i.e., loose contacts among side chains and lack of a specific hydrophobic core. The unfolded state is heterogeneous and includes a variety of α/β topologies but also fully α-helical and fully β-sheet structures. Transitions within the denatured state are very fast, and the molten-globule state is reached in <1 μs by a framework mechanism of folding with multiple pathways. The native structure of the wild-type is more rigid than the molten-globule conformation of the simplified-sequence variant. The difference in structural stability and the very fast folding of the simplified protein suggest that evolution has enriched the primordial alphabet of amino acids mainly to optimize protein function by stabilization of a unique structure with specific tertiary interactions