Approximating Projections of Conformational Boltzmann Distributions with AlphaFold2 Predictions: Opportunities and Limitations.

Protein thermodynamics is intimately tied to biological function and can enable processes such as signal transduction, enzyme catalysis, and molecular recognition. The relative free energies of conformations that contribute to these functional equilibria evolved for the physiology of the organism. Despite the importance of these equilibria for understanding biological function and developing treatments for disease, computational and experimental methods capable of quantifying the energetic determinants of these equilibria are limited to systems of modest size. Recently, it has been demonstrated that the artificial intelligence system AlphaFold2 can be manipulated to produce structurally valid protein conformational ensembles. Here, we extend these studies and explore the extent to which AlphaFold2 contact distance distributions can approximate projections of the conformational Boltzmann distributions. For this purpose, we examine the joint probability distributions of inter-residue contact distances along functionally relevant collective variables of several protein systems. Our studies suggest that AlphaFold2 normalized contact distance distributions can correlate with conformation probabilities obtained with other methods but that they suffer from peak broadening. We also find that the AlphaFold2 contact distance distributions can be sensitive to point mutations. Overall, we anticipate that our findings will be valuable as the community seeks to model the thermodynamics of conformational changes in large biomolecular systems.

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein glucose-6-phosphate catalytic subunit 1 (G6PC1) regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 causes glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. In this study, we determine the atomic interactions governing G6P binding as well as explore the perturbations imposed by disease-linked missense variants by subjecting an AlphaFold2 G6PC1 structural model to molecular dynamics simulations and in silico predictions of thermodynamic stability validated with robust in vitro and in situ biochemical assays. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. The introduction of GSD type 1a mutations modified the thermodynamic landscape, altered side chain packing and substrate-binding interactions, and induced trapping of catalytic intermediates. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm the active-site structural organization but also identify previously unobserved mechanistic contributions of catalytic and noncatalytic side chains.

Biofilme detectado em ponta de cateter venoso central por cultura usando método quantitativo / Biofilm detection on central venous catheter using a quantitative culture method

O cateter venoso central é um instrumento médico muito usado no monitoramento de pacientes em estado crítico. O uso destes cateteres expõe o paciente ao risco de infecções locais e sistêmicas. O objetivo deste estudo foi detectar a presença de microrganismosque colonizam cateteres venosos centrais usando método quantitativo de cultura. No período de um ano foram analisadas 118 pontas de cateter venoso central e 42 amostras de sangue de veia periférica colhidas de 100 pacientes internados em unidade deterapia intensiva de um hospital (idade média de 57,8±0,3). De um segmento de 2,0cm foi realizada uma cultura quantitativa usando a água Mili-Q que lavou a superfície interna do cateter, após agitação em vortex. De uma diluição 1/10, uma alíquota de 100μl foi semeada em placas de ágar sangue. As colônias foram relatadas como UFC/ml. Este método requer crescimento ≥103 UFC/ml para mostrar que o cateter esta colonizado. Os dados obtidos mostraram que 28,8% pontas estavam colonizadas. Foram isolados 33 cepas microbianasGram-positivas predominando S. aureus (30,7%), S. epidermidis (7,7%) e C. albicans (1,9%). Dezenove Gram-negativas, das quais A. baumannii (9,7%), E. aerogenes (5,8%), P. aeruginosa (3,9%) e S. maltophilia (3,9%) foram isolados. Das 42 culturas de sangue, quatro foram positivas e duas mostraram S. aureus, uma S. maltophilia e uma C. freundii. O perfil de suscetilidade de S. aureus mostrou múltipla resistência antimicrobiana aos aminoglicosídeos, cloranfenicol, lincosamidas, macrolídeos, penicilinas, quinolonas