A Perspective on the Prospective Use of AI in Protein Structure Prediction.

AlphaFold2 (AF2) and RoseTTaFold (RF) have revolutionized structural biology, serving as highly reliable and effective methods for predicting protein structures. This article explores their impact and limitations, focusing on their integration into experimental pipelines and their application in diverse protein classes, including membrane proteins, intrinsically disordered proteins (IDPs), and oligomers. In experimental pipelines, AF2 models help X-ray crystallography in resolving the phase problem, while complementarity with mass spectrometry and NMR data enhances structure determination and protein flexibility prediction. Predicting the structure of membrane proteins remains challenging for both AF2 and RF due to difficulties in capturing conformational ensembles and interactions with the membrane. Improvements in incorporating membrane-specific features and predicting the structural effect of mutations are crucial. For intrinsically disordered proteins, AF2's confidence score (pLDDT) serves as a competitive disorder predictor, but integrative approaches including molecular dynamics (MD) simulations or hydrophobic cluster analyses are advocated for accurate dynamics representation. AF2 and RF show promising results for oligomeric models, outperforming traditional docking methods, with AlphaFold-Multimer showing improved performance. However, some caveats remain in particular for membrane proteins. Real-life examples demonstrate AF2's predictive capabilities in unknown protein structures, but models should be evaluated for their agreement with experimental data. Furthermore, AF2 models can be used complementarily with MD simulations. In this Perspective, we propose a "wish list" for improving deep-learning-based protein folding prediction models, including using experimental data as constraints and modifying models with binding partners or post-translational modifications. Additionally, a meta-tool for ranking and suggesting composite models is suggested, driving future advancements in this rapidly evolving field.

Inverse Conformational Selection in Lipid-Protein Binding.

Interest in lipid interactions with proteins and other biomolecules is emerging not only in fundamental biochemistry but also in the field of nanobiotechnology where lipids are commonly used, for example, in carriers of mRNA vaccines. The outward-facing components of cellular membranes and lipid nanoparticles, the lipid headgroups, regulate membrane interactions with approaching substances, such as proteins, drugs, RNA, or viruses. Because lipid headgroup conformational ensembles have not been experimentally determined in physiologically relevant conditions, an essential question about their interactions with other biomolecules remains unanswered: Do headgroups exchange between a few rigid structures, or fluctuate freely across a practically continuous spectrum of conformations? Here, we combine solid-state NMR experiments and molecular dynamics simulations from the NMRlipids Project to resolve the conformational ensembles of headgroups of four key lipid types in various biologically relevant conditions. We find that lipid headgroups sample a wide range of overlapping conformations in both neutral and charged cellular membranes, and that differences in the headgroup chemistry manifest only in probability distributions of conformations. Furthermore, the analysis of 894 protein-bound lipid structures from the Protein Data Bank suggests that lipids can bind to proteins in a wide range of conformations, which are not limited by the headgroup chemistry. We propose that lipids can select a suitable headgroup conformation from the wide range available to them to fit the various binding sites in proteins. The proposed inverse conformational selection model will extend also to lipid binding to targets other than proteins, such as drugs, RNA, and viruses.

Transceive phase corrected 2D contrast source inversion-electrical properties tomography.

PURPOSE: To remove the necessity of the tranceive phase assumption for CSI-EPT and show electrical properties maps reconstructed from measured data obtained using a standard 3T birdcage body coil setup. METHODS: The existing CSI-EPT algorithm is reformulated to use the transceive phase rather than relying on the transceive phase assumption. Furthermore, the radio frequency (RF)-shield is numerically implemented to accurately model the RF fields inside the MRI scanner. We verify that the reformulated two-dimensional (2D) CSI-EPT algorithm can reconstruct electrical properties maps given 2D electromagnetic simulations. Afterward, the algorithm is tested with three-dimensional (3D) FDTD simulations to investigate if the 2D CSI-EPT can retrieve the electrical properties for 3D RF fields. Finally, an MR experiment at 3T with a phantom is performed. RESULTS: From the results of the 2D simulations, it is seen that CSI-EPT can reconstruct the electrical properties using MRI accessible quantities. For 3D simulations, it is observed that the electrical properties are underestimated, nonetheless, CSI-EPT has a lower standard deviation than the standard Helmholtz based methods. Finally, the first CSI-EPT reconstructions based on measured data are presented showing comparable accuracy and precision to reconstructions based on simulated data, and demonstrating the feasibility of CSI-EPT. CONCLUSIONS: The CSI-EPT algorithm was rewritten to use MRI accessible quantities. This allows for CSI-EPT to fully exploit the benefits of the higher static magnetic field strengths with a standard quadrature birdcage coil setup.

On the Effect of the Various Assumptions and Approximations used in Molecular Simulations on the Properties of Bio-Molecular Systems: Overview and Perspective on Issues.

Computer simulations of molecular systems enable structure-energy-function relationships of molecular processes to be described at the sub-atomic, atomic, supra-atomic or supra-molecular level and plays an increasingly important role in chemistry, biology and physics. To interpret the results of such simulations appropriately, the degree of uncertainty and potential errors affecting the calculated properties must be considered. Uncertainty and errors arise from (1) assumptions underlying the molecular model, force field and simulation algorithms, (2) approximations implicit in the interatomic interaction function (force field), or when integrating the equations of motion, (3) the chosen values of the parameters that determine the accuracy of the approximations used, and (4) the nature of the system and the property of interest. In this overview, advantages and shortcomings of assumptions and approximations commonly used when simulating bio-molecular systems are considered. What the developers of bio-molecular force fields and simulation software can do to facilitate and broaden research involving bio-molecular simulations is also discussed.

Molecular Study of Ultrasound-Triggered Release of Fluorescein from Liposomes.

Several investigations have suggested that ultrasound triggers the release of drugs encapsulated into liposomes at acoustic pressures low enough to avoid cavitation or high hyperthermia. However, the mechanism leading to this triggered release as well as the adequate composition of the liposome membrane remains unknown. Here, we investigate the ultrasound-triggered release of fluorescein disodium salt encapsulated into liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-distearoylphosphatidyl-ethanolamine (DSPC) lipids with various concentrations of cholesterol (from 0 to 44 mol %). The passive release of encapsulated fluorescein was first characterized. It was observed to be higher when the membrane is in a fluid phase and increased with temperature but decreased upon addition of cholesterol. Next, the release of fluorescein was measured at different acoustic frequencies (0.8, 1.1, and 3.3 MHz) and peak-to-peak pressures (0, 2, 2.5, 5, and 8 MPa). Measurements were performed at temperatures where DOPC and DSPC liposomes were, respectively, in the fluid or gel phase. We found that the release rate did not depend on the ultrasound frequency. For DOPC liposomes, the ultrasound-triggered release of fluorescein decreased with increasing concentration of cholesterol in liposomes, while the behavior was more complex for DSPC liposomes. Overall, the triggered release from DSPC liposomes was up to ten times less than DOPC liposomes. Molecular dynamics simulations performed on a pure DOPC membrane showed that a membrane experiences, under a directional pressure of ±2.4 MPa, various changes in properties such as the area per lipid (APL). An increase in the APL was notably observed when the simulation box was laterally stretched or perpendicularly compressed, which was accompanied by an increase in the number of water molecules crossing the membrane. This suggests that ultrasound most probably enhances the diffusion of encapsulated molecules at small acoustic pressures by increasing the distance between lipids.

The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion.

Mitochondria are double-membrane-bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane-bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C-terminal amphipathic tail of the Atlastin protein.

Meet-U: Educating through research immersion.

We present a new educational initiative called Meet-U that aims to train students for collaborative work in computational biology and to bridge the gap between education and research. Meet-U mimics the setup of collaborative research projects and takes advantage of the most popular tools for collaborative work and of cloud computing. Students are grouped in teams of 4-5 people and have to realize a project from A to Z that answers a challenging question in biology. Meet-U promotes "coopetition," as the students collaborate within and across the teams and are also in competition with each other to develop the best final product. Meet-U fosters interactions between different actors of education and research through the organization of a meeting day, open to everyone, where the students present their work to a jury of researchers and jury members give research seminars. This very unique combination of education and research is strongly motivating for the students and provides a formidable opportunity for a scientific community to unite and increase its visibility. We report on our experience with Meet-U in two French universities with master's students in bioinformatics and modeling, with protein-protein docking as the subject of the course. Meet-U is easy to implement and can be straightforwardly transferred to other fields and/or universities. All the information and data are available at www.meet-u.org.

Biophysical Insight on the Membrane Insertion of an Arginine-Rich Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs) are short peptides that can translocate and transport cargoes into the intracellular milieu by crossing biological membranes. The mode of interaction and internalization of cell-penetrating peptides has long been controversial. While their interaction with anionic membranes is quite well understood, the insertion and behavior of CPPs in zwitterionic membranes, a major lipid component of eukaryotic cell membranes, is poorly studied. Herein, we investigated the membrane insertion of RW16 into zwitterionic membranes, a versatile CPP that also presents antibacterial and antitumor activities. Using complementary approaches, including NMR spectroscopy, fluorescence spectroscopy, circular dichroism, and molecular dynamic simulations, we determined the high-resolution structure of RW16 and measured its membrane insertion and orientation properties into zwitterionic membranes. Altogether, these results contribute to explaining the versatile properties of this peptide toward zwitterionic lipids.

PackMem: A Versatile Tool to Compute and Visualize Interfacial Packing Defects in Lipid Bilayers.

The analysis of the structural organization of lipid bilayers is generally performed across the direction normal to the bilayer/water interface, whereas the surface properties of the bilayer at the interface with water are often neglected. Here, we present PackMem, a bioinformatic tool that performs a topographic analysis of the bilayer surface from various molecular dynamics simulations. PackMem unifies and rationalizes previous analyses based on a Cartesian grid. The grid allows identification of surface regions defined as lipid-packing defects where lipids are loosely packed, leading to cavities in which aliphatic carbons are exposed to the solvent, either deep inside or close to the membrane surface. Examples are provided to show that the abundance of lipid-packing defects varies according to the temperature and to the bilayer composition. Because lipid-packing defects control the adsorption of peripheral proteins with hydrophobic insertions, PackMem is instrumental for us to understand and quantify the adhesive properties of biological membranes as well as their response to mechanical perturbations such as membrane deformation.

Targeting surface voids to counter membrane disorders in lipointoxication-related diseases.

Saturated fatty acids (SFA), which are abundant in the so-called western diet, have been shown to efficiently incorporate within membrane phospholipids and therefore impact on organelle integrity and function in many cell types. In the present study, we have developed a yeast-based two-step assay and a virtual screening strategy to identify new drugs able to counter SFA-mediated lipointoxication. The compounds identified here were effective in relieving lipointoxication in mammalian ß-cells, one of the main targets of SFA toxicity in humans. In vitro reconstitutions and molecular dynamics simulations on bilayers revealed that these molecules, albeit according to different mechanisms, can generate voids at the membrane surface. The resulting surface defects correlate with the recruitment of loose lipid packing or void-sensing proteins required for vesicular budding, a central cellular process that is precluded under SFA accumulation. Taken together, the results presented here point at modulation of surface voids as a central parameter to consider in order to counter the impacts of SFA on cell function.

Interdigitation between Triglycerides and Lipids Modulates Surface Properties of Lipid Droplets.

Intracellular lipid droplets (LDs) are the main cellular site of metabolic energy storage. Their structure is unique inside the cell, with a core of esterified fatty acids and sterols, mainly triglycerides and sterol esters, surrounded by a single monolayer of phospholipids. Numerous peripheral proteins, including several that were previously associated with intracellular compartments surrounded by a lipid bilayer, have been recently shown to target the surface of LDs, but how they are able to selectively target this organelle remains largely unknown. Here, we use atomistic and coarse-grained molecular dynamics simulations to investigate the molecular properties of the LD surface and to characterize how it differs from that of a lipid bilayer. Our data suggest that although several surface properties are remarkably similar between the two structures, key differences originate from the interdigitation between surface phospholipids and core neutral lipids that occurs in LDs. This property is extremely sensitive to membrane undulations, unlike in lipid bilayers, and it strongly affects both lipid-packing defects and the lateral pressure profile. We observed a marked change in overall surface properties for surface tensions >10 mN/m, indicative of a bimodal behavior. Our simulations provide a comprehensive molecular characterization of the unique surface properties of LDs and suggest how the molecular properties of the surface lipid monolayer can be modulated by the underlying neutral lipids.

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Simulating Bilayers of Nonionic Surfactants with the GROMOS-Compatible 2016H66 Force Field.

Polyoxyethylene glycol alkyl ether amphiphiles (CiEj) are important nonionic surfactants, often used for biophysical and membrane protein studies. In this work, we extensively test the GROMOS-compatible 2016H66 force field in molecular dynamics simulations involving the lamellar phase of a series of CiEj surfactants, namely C12E2, C12E3, C12E4, C12E5, and C14E4. The simulations reproduce qualitatively well the monitored structural properties and their experimental trends along the surfactant series, although some discrepancies remain, in particular in terms of the area per surfactant, the equilibrium phase of C12E5, and the order parameters of C12E3, C12E4, and C12E5. The polar head of the CiEj surfactants is highly hydrated, almost like a single polyethyleneoxide (PEO) molecule at full hydration, resulting in very compact conformations. Within the bilayer, all CiEj surfactants flip-flop spontaneously within tens of nanoseconds. Water-permeation is facilitated, and the bending rigidity is 4 to 5 times lower than that of typical phospholipid bilayers. In line with another recent theoretical study, the simulations show that the lamellar phase of CiEj contains large hydrophilic pores. These pores should be abundant in order to reproduce the comparatively low NMR order parameters. We show that their contour length is directly correlated to the order parameters, and we estimate that they should occupy approximately 7-10% of the total membrane area. Due to their highly dynamic nature (rapid flip-flops, high water permeability, observed pore formation), CiEj surfactant bilayers are found to represent surprisingly challenging systems in terms of modeling. Given this difficulty, the results presented here show that the 2016H66 parameters, optimized independently considering pure-liquid as well as polar and nonpolar solvation properties of small organic molecules, represent a good starting point for simulating these systems.

Nitroalkenes as Latent 1,2-Biselectrophiles - A Multicatalytic Approach for the Synthesis of 1,4-Diketones and Their Application in a Four-Step One-Pot Reaction to Polysubstituted Pyrroles.

An NHC-catalyzed nitro-Stetter/elimination/Stetter reaction sequence employs nitroalkenes as latent 1,2-dication synthons providing a novel access to highly useful symmetrical and unsymmetrical 2-aryl substituted 1,4-diketone building blocks from commercially available aldehyde precursors. For less activated (aliphatic) aldehydes, a cooperative catalytic strategy has been developed via the merger of NHC and H-bonding catalysis. To further showcase the versatility of our approach, a great variety of these unprecedented 1,4-diketones are used to efficiently synthesize polysubstituted pyrroles-including those with hetaryl substituents-in good to excellent yields in a multicatalytic metal-free, four-step one-pot cascade reaction under mild, yet robust, conditions.

Remote-Stereocontrol in Dienamine Catalysis: Z-Dienamine Preferences and Electrophile-Catalyst Interaction Revealed by NMR and Computational Studies.

Catalysis with remote-stereocontrol provides special challenges in design and comprehension. One famous example is the dienamine catalysis, for which high ee values are reported despite insufficient shielding of the second double bond. Especially for dienamines with variable Z/E-ratios of the second double bond, no correlations to the ee values are found. Therefore, the structures, thermodynamics, and kinetics of dienamine intermediates in SN-type reactions are investigated. The NMR studies show that the preferred dienamine conformation provides an effective shielding if large electrophiles are used. Calculations at SCS-MP2/CBS-level of theory and experimental data of the dienamine formation show kinetic preference for the Z-isomer of the second double bond and a slow isomerization toward the thermodynamically preferred E-isomer. Modulations of the rate-determining step, by variation of the concentration of the electrophile, allow the conversion of dienamines to be observed. With electrophiles, a faster reaction of Z- than of E-isomers is observed experimentally. Calculations corroborate these results by correlating ee values of three catalysts with the kinetics of the electrophilic attack and reveal the significance of CH-π and stacking interactions in the transition states. Thus, for the first time a comprehensive understanding of the remote stereocontrol in γ-functionalization reactions of dienamines and an explanation to the "Z/E-dilemma" are presented. The combination of bulky catalyst subsystems and large electrophiles provides a shielding of one face and causes different reactivities of E/Z-dienamines in nucleophilic attacks from the other face. Kinetic preferences for the formation of Z-dienamines and their unfavorable thermodynamics support high ee values.

Coarse-grained simulations of the HIV-1 matrix protein anchoring: revisiting its assembly on membrane domains.

In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ~5 kcal.mol(-1). The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2' acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P2 lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P2 in domains. This is consistent with a PI(4,5)P2 enrichment of the virus envelope as compared to the host cell membrane.