Fast Folding Research Articles

Impact of Hydrodynamic Interactions on the Kinetic Pathway of Protein Folding.

Protein folding is a fundamental process critical to cellular function and human health, but it remains a grand challenge in biophysics. Hydrodynamic interaction (HI) plays a vital role in the self-organization of soft and biological materials, yet its role in protein folding is not fully understood despite folding occurring in a fluid environment. Here, we use the fluid particle dynamics method to investigate many-body hydrodynamic couplings between amino acid residues and fluid motion in the folding kinetics of a coarse-grained four-α-helices bundle protein. Our results reveal that HI helps select fast folding pathways to the native state without being kinetically trapped, significantly speeding up the folding kinetics compared to its absence. First, the directional flow along the protein backbone expedites protein collapse. Then, the incompressibility-induced squeezing flow effects retard the accumulation of non-native hydrophobic contacts, thus preventing the protein from being trapped in local energy minima during the conformational search of the native structure. We also find that the significance of HI in folding kinetics depends on temperature, with a pronounced effect under biologically relevant conditions. Our findings suggest that HI, particularly the short-range squeezing effect, may be crucial in avoiding protein misfolding.

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics.

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

Early formed non-local contacts are “nature’s shortcuts” to fast protein folding

Early formed non-local contacts are “nature’s shortcuts” to fast protein folding

A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Multi-eGO: Model Improvements toward the Study of Complex Self-Assembly Processes.

Structure-based models have been instrumental in simulating protein folding and suggesting hypotheses about the mechanisms involved. Nowadays, at least for fast-folding proteins, folding can be simulated in explicit solvent using classical molecular dynamics. However, other self-assembly processes, such as protein aggregation, are still far from being accessible. Recently, we proposed that a hybrid multistate structure-based model, multi-eGO, could help to bridge the gap toward the simulation of out-of-equilibrium, concentration-dependent self-assembly processes. Here, we further improve the model and show how multi-eGO can effectively and accurately learn the conformational ensemble of the amyloid β42 intrinsically disordered peptide, reproduce the well-established folding mechanism of the B1 immunoglobulin-binding domain of streptococcal protein G, and reproduce the aggregation as a function of the concentration of the transthyretin 105-115 amyloidogenic peptide. We envision that by learning from the dynamics of a few minima, multi-eGO can become a platform for simulating processes inaccessible to other simulation techniques.

A Search for Pulsars around Sgr A* in the First Event Horizon Telescope Data Set

In 2017 the Event Horizon Telescope (EHT) observed the supermassive black hole at the center of the Milky Way, Sagittarius A* (Sgr A*), at a frequency of 228.1 GHz (λ = 1.3 mm). The fundamental physics tests that even a single pulsar orbiting Sgr A* would enable motivate searching for pulsars in EHT data sets. The high observing frequency means that pulsars—which typically exhibit steep emission spectra—are expected to be very faint. However, it also negates pulse scattering, an effect that could hinder pulsar detections in the Galactic center. Additionally, magnetars or a secondary inverse Compton emission could be stronger at millimeter wavelengths than at lower frequencies. We present a search for pulsars close to Sgr A* using the data from the three most sensitive stations in the EHT 2017 campaign: the Atacama Large Millimeter/submillimeter Array, the Large Millimeter Telescope, and the IRAM 30 m Telescope. We apply three detection methods based on Fourier-domain analysis, the fast folding algorithm, and single-pulse searches targeting both pulsars and burst-like transient emission. We use the simultaneity of the observations to confirm potential candidates. No new pulsars or significant bursts were found. Being the first pulsar search ever carried out at such high radio frequencies, we detail our analysis methods and give a detailed estimation of the sensitivity of the search. We conclude that the EHT 2017 observations are only sensitive to a small fraction (≲2.2%) of the pulsars that may exist close to Sgr A*, motivating further searches for fainter pulsars in the region.

The High Time Resolution Universe Pulsar survey – XVIII. The reprocessing of the HTRU-S Low Lat survey around the Galactic Centre using a Fast Folding Algorithm pipeline for accelerated pulsars

ABSTRACT The HTRU-S Low Latitude survey data within 1° of the Galactic Centre (GC) were searched for pulsars using the Fast Folding Algorithm (FFA). Unlike traditional Fast Fourier Transform (FFT) pipelines, the FFA optimally folds the data for all possible periods over a given range, which is particularly advantageous for pulsars with low-duty cycles. For the first time, a search over acceleration was included in the FFA to improve its sensitivity to binary pulsars. The steps in dispersion measure (DM) and acceleration were optimized, resulting in a reduction of the number of trials by 86 per cent. This was achieved over a search period range from 0.6 to 432-s, i.e. 10 per cent of the observation time (4320s), with a maximum DM of 4000 pc cm−3 and an acceleration range of ±128 m s−2. The search resulted in the re-detections of four known pulsars, including a pulsar that was missed in the previous FFT processing of this survey. This result indicates that the FFA pipeline is more sensitive than the FFT pipeline used in the previous processing of the survey within our parameter range. Additionally, we discovered a 1.89-s pulsar, PSR J1746-2829, with a large DM, located 0.5 from the GC. Follow-up observations revealed that this pulsar has a relatively flat spectrum (α = −0.9 ± 0.1) and has a period derivative of ∼1.3 × 10−12 s s−1, implying a surface magnetic field of ∼5.2 × 1013 G and a characteristic age of ∼23 000 yr. While the period, spectral index, and surface magnetic field strength are similar to many radio magnetars, other characteristics such as high linear polarization are absent.

In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non-native interactions.

We examine the influence of cellular interactions in all-atom models of a section of the Homo sapiens cytoplasm on the early folding events of the three-helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30-microsecond simulations. We did however capture the formation of all three helices in a compact native-like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta-sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild-type sequence. This study demonstrates that even fast-folding proteins can get stuck in non-native states in the cell, making them useful models for protein-chaperone interactions and early stages of aggregate formation relevant to cellular disease.

Measuring protein unfolding thermodynamic stability in one minute with digital temperature control-equipped nanoESI-mass spectrometry

Rapidly measuring protein thermodynamic stability is vital for understanding the fast protein folding and unfolding processes, involving many human disease-linked protein structures and the fulfilment of their dynamic functions. Heating-induced protein unfolding and conformational transformation studies may generate a variety of protein thermodynamic stability information such as protein melting temperature (Tm) and Gibbs free energy (ΔG). Previous nanoelectrospray ionization (nanoESI) - mass spectrometry (MS) has been interfaced with online heating device to achieve such information, but mostly operating in a slow mode in terms of temperature control. Herein, a new module for digital temperature control (DTC) was constructed and assembled into a nanoESI device, allowing for ultrafast measurement of thermodynamic stability. Typically, DTC can achieve whole-range heating-induced unfolding in 33 s with temperatures ranging from 0 °C to 99 °C but with jump step of 3 °C and deviation less than 1 °C. Notably, thanks to the advantage of ultrafast and precise temperature control, only less than 100 nL protein sample was consumed for each test, saving samples by more than 100 folds compared to previously reported temperature control devices. Besides, with the DTC-nanoESI-MS regime, we successfully achieved solution pH-dependent protein thermodynamics, which serves as first proof-of-concept demonstration for future applications even at a proteome level directly from limited biological samples.

The GMRT High Resolution Southern Sky Survey for Pulsars and Transients. VI. Discovery of Nulling, Localization, and Timing of PSR J1244–4708

Many pulsars in the known population exhibit nulling, which is characterized by a sudden cessation and subsequent restoration of radio emission. In this work, we present the localization, timing, and emission properties of a pulsar discovered by the GMRT High Resolution Southern Sky survey: J1244–4708. Moreover, we find that this pulsar shows nulling, with a nulling fraction close to 60%. A quasiperiodicity is also seen in the nulling from this pulsar with two timescales. We demonstrate the broadband nature of nulling in this pulsar using simultaneous observations in band-3 (300–500 MHz) and band-4 (550–750 MHz) with the upgraded GMRT. We also present a comparison of the efficiency of various search approaches such as single pulse search, fast folding algorithm (FFA)–based search, and fast Fourier transform–based search to search for nulling pulsars. We demonstrated that the FFA search is advantageous for detecting extreme nulling pulsars, which is also confirmed with multiple epochs of observations for the nulling pulsars using the GMRT.

Fast protein folding is governed by memory-dependent friction

When described by a low-dimensional reaction coordinate, the folding rates of most proteins are determined by a subtle interplay between free-energy barriers, which separate folded and unfolded states, and friction. While it is commonplace to extract free-energy profiles from molecular trajectories, a direct evaluation of friction is far more elusive and typically relies on fits of measured reaction rates to memoryless reaction-rate theories. Here, using memory-kernel extraction methods founded on a generalized Langevin equation (GLE) formalism, we directly calculate the time-dependent friction acting on the fraction of native contacts reaction coordinate Q, evaluated for eight fast-folding proteins, taken from a published set of large-scale molecular dynamics protein simulations. Our results reveal that, across the diverse range of proteins represented in this dataset, friction is more influential than free-energy barriers in determining protein folding rates. We also show that proteins fold in a regime where the finite decay time of friction significantly reduces the folding times, in some instances by as much as a factor of 10, compared to predictions based on memoryless friction.

Design and analysis of an inflatable recovery platform for shipborne UAV

It is difficult to recover drones for small ships (such as destroyers). Therefore, it is necessary to design a new carrier-borne UAV recovery platform, achieving the purpose of rapid recovery of offshore UAV. This paper presents a fast unfolding and folding inflatable structure for small ship UAV offshore recovery. This paper analyze some static response of the UAV recovery platform. Firstly, parking positions of UAV on the runway surface is a factor. Secondly, runway surface materials is also a factor. Thirdly, airbag’s internal pressure also affects static response of the platform. It is find that static responses such as deformation and stress more sensitive to the runway surface material and the position of UAV, and the transverse deformation is sensitive to the runway surface material, but not to the internal pressure of the airbag.

A 4–8 GHz Galactic Center Search for Periodic Technosignatures

Radio searches for extraterrestrial intelligence have mainly targeted the discovery of narrowband continuous-wave beacons and artificially dispersed broadband bursts. Periodic pulse trains, in comparison to the above technosignature morphologies, offer an energetically efficient means of interstellar transmission. A rotating beacon at the Galactic Center (GC), in particular, would be highly advantageous for galaxy-wide communications. Here, we present blipss, a CPU-based open-source software that uses a fast folding algorithm (FFA) to uncover channel-wide periodic signals in radio dynamic spectra. Running blipss on 4.5 hr of 4–8 GHz data gathered with the Robert C. Byrd Green Bank Telescope, we searched the central of our galaxy for kHz-wide signals with periods between 11 and 100 s and duty cycles (δ) between 10% and 50%. Our searches, to our knowledge, constitute the first FFA exploration for periodic alien technosignatures. We report a nondetection of channel-wide periodic signals in our data. Thus, we constrain the abundance of 4–8 GHz extraterrestrial transmitters of kHz-wide periodic pulsed signals to fewer than one in about 600,000 stars at the GC above a 7σ equivalent isotropic radiated power of ≈2 × 1018 W at δ ≃ 10%. From an astrophysics standpoint, blipss, with its utilization of a per-channel FFA, can enable the discovery of signals with exotic radio frequency sweeps departing from the standard cold plasma dispersion law.

Deep Boosted Molecular Dynamics: Accelerating Molecular Simulations with Gaussian Boost Potentials Generated Using Probabilistic Bayesian Deep Neural Network.

We have developed a new deep boosted molecular dynamics (DBMD) method. Probabilistic Bayesian neural network models were implemented to construct boost potentials that exhibit Gaussian distribution with minimized anharmonicity, thereby allowing for accurate energetic reweighting and enhanced sampling of molecular simulations. DBMD was demonstrated on model systems of alanine dipeptide and the fast-folding protein and RNA structures. For alanine dipeptide, 30 ns DBMD simulations captured up to 83-125 times more backbone dihedral transitions than 1 μs conventional molecular dynamics (cMD) simulations and were able to accurately reproduce the original free energy profiles. Moreover, DBMD sampled multiple folding and unfolding events within 300 ns simulations of the chignolin model protein and identified low-energy conformational states comparable to previous simulation findings. Finally, DBMD captured a general folding pathway of three hairpin RNAs with the GCAA, GAAA, and UUCG tetraloops. Based on a deep learning neural network, DBMD provides a powerful and generally applicable approach to boosting biomolecular simulations. DBMD is available with open source in OpenMM at https://github.com/MiaoLab20/DBMD/.

Visualizing the Residue Interaction Landscape of Proteins by Temporal Network Embedding.

Characterizing the structural dynamics of proteins with heterogeneous conformational landscapes is crucial to understanding complex biomolecular processes. To this end, dimensionality reduction algorithms are used to produce low-dimensional embeddings of the high-dimensional conformational phase space. However, identifying a compact and informative set of input features for the embedding remains an ongoing challenge. Here, we propose to harness the power of Residue Interaction Networks (RINs) and their centrality measures, established tools to provide a graph theoretical view on molecular structure. Specifically, we combine the closeness centrality, which captures global features of the protein conformation at residue-wise resolution, with EncoderMap, a hybrid neural-network autoencoder/multidimensional-scaling like dimensionality reduction algorithm. We find that the resulting low-dimensional embedding is a meaningful visualization of the residue interaction landscape that resolves structural details of the protein behavior while retaining global interpretability. This feature-based graph embedding of temporal protein graphs makes it possible to apply the general descriptive power of RIN formalisms to the analysis of protein simulations of complex processes such as protein folding and multidomain interactions requiring no protein-specific input. We demonstrate this on simulations of the fast folding protein Trp-Cage and the multidomain signaling protein FAT10. Due to its generality and modularity, the presented approach can easily be transferred to other protein systems.

Predicting residue cooperativity during protein folding: A combined, molecular dynamics and unsupervised learning approach.

Allostery in proteins involves, broadly speaking, ligand-induced conformational transitions that modulate function at active sites distal to where the ligand binds. In contrast, the concept of cooperativity (in the sense used in phase transition theory) is often invoked to understand protein folding and, therefore, function. The modern view on allostery is one based on dynamics and hinges on the time-dependent interactions between key residues in a complex network, interactions that determine the free-energy profile for the reaction at the distal site. Here, we merge allostery and cooperativity, and we discuss a joint model with features of both. In our model, the active-site reaction is replaced by the reaction pathway that leads to protein folding, and the presence or absence of the effector is replaced by mutant-vs-wild type changes in key residues. To this end, we employ our recently introduced time-lagged independent component analysis (tICA) correlation approach [Ray et al. Proc. Natl. Acad. Sci. 118(43) (2021), e2100943118] to identify the allosteric role of distant residues in the folded-state dynamics of a large protein. In this work, we apply the technique to identify key residues that have a significant role in the folding of a small, fast folding-protein, chignolin. Using extensive enhanced sampling simulations, we critically evaluate the accuracy of the predictions by mutating each residue one at a time and studying how the mutations change the underlying free energy landscape of the folding process. We observe that mutations in those residues whose associated backbone torsion angles have a high correlation score can indeed lead to loss of stability of the folded configuration. We also provide a rationale based on interaction energies between individual residues with the rest of the protein to explain this effect. From these observations, we conclude that the tICA correlation score metric is a useful tool for predicting the role of individual residues in the correlated dynamics of proteins and can find application to the problem of identifying regions of protein that are either most vulnerable to mutations or-mutatis mutandis-to binding events that affect their functionality.

TMAO: Protecting proteins from feeling the heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 μs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules “herding” water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

The GMRT High Resolution Southern Sky Survey for Pulsars and Transients. IV. Discovery of Four New Pulsars with an FFA Search

The fast Fourier transform (FFT) based periodicity search methods provide an efficient way to search for millisecond and binary pulsars but encounter significant sensitivity degradation while searching for long period and short duty cycle pulsars. An alternative to FFT-based search methods called the fast folding algorithm (FFA) search provides superior sensitivity to search for signals with long periods and short duty cycles. In the GMRT High Resolution Southern Sky (GHRSS) survey, we are using an FFA-based pipeline to search for isolated pulsars in a period range of 100 ms to 100 s. We have processed 2800 degree2 of the sky coverage away from the Galactic plane and discovered six new pulsars. Here, we report the discovery of four of these pulsars with the FFA search pipeline. This includes a narrow duty cycle pulsar, J1936−30, which shows nulling behavior with an extreme nulling fraction of ∼90%. Two of the GHRSS discoveries from the FFA search lie in narrow duty cycle ranges beyond the limit of the existing population. The implementation of FFA search in the GHRSS survey and other pulsar surveys is expected to recover the missing population of long period and short duty cycle pulsars.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

The Role of Transient Intermediate Structures in the Unfolding of the Trp-Cage Fast-Folding Protein: Generating Ensembles from Time-Resolved X-ray Solution Scattering with Genetic Algorithms.

The Trp-cage miniprotein is one of the smallest systems to exhibit a stable secondary structure and fast-folding dynamics, serving as an apt model system to study transient intermediates with both experimental and computational analyses. Previous spectroscopic characterizations that have been done on Trp-cage have inferred a single stable intermediate on a pathway from folded to unfolded basins. We aim to bridge the understanding of Trp-cage structural folding dynamics on microsecond-time scales, by utilizing time-resolved X-ray solution scattering to probe the temperature-induced unfolding pathway. Our results indicate the formation of a conformationally extended intermediate on the time scale of 1 μs, which undergoes complete unfolding within 5 μs. We further investigated the atomistic structural details of the unfolding pathway using a genetic algorithm to generate ensemble model fits to the scattering profiles. This analysis paves the way for direct benchmarking of theoretical models of protein folding ensembles produced with molecular dynamics simulations.