Background: Proteins fold robustly and reproducibly in vivo, but many cannot fold in vitro in isolation from cellular components. Despite the remarkable progress that has been achieved by the artificial intelligence approaches in predicting the protein native conformations, the pathways that lead to such conformations, either in vitro or in vivo, remain largely unknown. The slow progress in recapitulating protein folding pathways in silico may be an indication of the fundamental deficiencies in our understanding of folding as it occurs in nature. Here we consider the possibility that protein folding in living cells may not be driven solely by the decrease in Gibbs free energy and propose that protein folding in vivo should be modeled as an active energy-dependent process. The mechanism of action of such a protein folding machine might include direct manipulation of the peptide backbone. Methods: To show the feasibility of a protein folding machine, we conducted molecular dynamics simulations that were augmented by the application of mechanical force to rotate the C-terminal amino acid while simultaneously limiting the N-terminal amino acid movements. Results: Remarkably, the addition of this simple manipulation of peptide backbones to the standard molecular dynamics simulation indeed facilitated the formation of native structures in five diverse alpha-helical peptides. Steric clashes that arise in the peptides due to the forced directional rotation resulted in the behavior of the peptide backbone no longer resembling a freely jointed chain. Conclusions: These simulations show the feasibility of a protein folding machine operating under the conditions when the movements of the polypeptide backbone are restricted by applying external forces and constraints. Further investigation is needed to see whether such an effect may play a role during co-translational protein folding in vivo and how it can be utilized to facilitate folding of proteins in artificial environments.

Nature has shaped the make up of proteins since their appearance, [Formula: see text]3.8 billion years ago. However, the fundamental drivers of structural change responsible for the extraordinary diversity of proteins have yet to be elucidated. Here we explore if protein evolution affects folding speed. We estimated folding times for the present-day catalog of protein domains directly from their size-modified contact order. These values were mapped onto an evolutionary timeline of domain appearance derived from a phylogenomic analysis of protein domains in 989 fully-sequenced genomes. Our results show a clear overall increase of folding speed during evolution, with known ultra-fast downhill folders appearing rather late in the timeline. Remarkably, folding optimization depends on secondary structure. While alpha-folds showed a tendency to fold faster throughout evolution, beta-folds exhibited a trend of folding time increase during the last [Formula: see text]1.5 billion years that began during the "big bang" of domain combinations. As a consequence, these domain structures are on average slow folders today. Our results suggest that fast and efficient folding of domains shaped the universe of protein structure. This finding supports the hypothesis that optimization of the kinetic and thermodynamic accessibility of the native fold reduces protein aggregation propensities that hamper cellular functions.

The Hsp70 chaperone system is the major molecular chaperone system that assists protein-folding processes in all cells. To understand these processes, we analyzed the kinetic characteristics of the Escherichia coli homologs of this chaperone system during folding of a denatured protein using computer simulations and compared the results with in vitro refolding experiments. Rate constants used for the model were derived from recent literature or were determined and scrutinized for their applicability to the refolding reaction. Our simulation results are consistent with reported laboratory experiments, not only simulating the refolding reaction of wild-type proteins but also the behavior of mutant variants. Variation of kinetic parameters and concentrations of components of the Hsp70 system demonstrate the robustness of the chaperone system in assisting protein folding. Furthermore, the importance of the synergistic stimulation of the ATPase activity of Hsp70 is demonstrated. The limitations of our kinetic model indicate sore spots in our understanding of this chaperone system. Our model provides a platform for further research on chaperone action and the mechanism of chaperone-assisted refolding of denatured proteins.

Maintaining the health of the proteome is a critical cellular task. Recently, we found G-quadruplex (G4) nucleic acids are especially potent at preventing protein aggregation in vitro and could at least indirectly improve the protein folding environment of Escherichia coli. However, the roles of G4s in protein folding were not yet explored. Here, through in vitro protein folding experiments, we discover that G4s can accelerate protein folding by rescuing kinetically trapped intermediates to both native and near-native folded states. Time-course folding experiments in E. coli further demonstrate that these G4s primarily improve protein folding quality in E. coli as opposed to preventing protein aggregation. The ability of a short nucleic acid to rescue protein folding opens up the possibility of nucleic acids and ATP-independent chaperones to play considerable roles in dictating the ultimate folding fate of proteins.

Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone-substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.

A diffusion theory-based, all-physical ab initio protein folding simulation is described and applied. The model is based upon the drift and diffusion of protein substructures relative to one another in the multiple energy fields present. Without templates or statistical inputs, the simulations were run at physiologic and ambient temperatures (including pH). Around 100 protein secondary structures were surveyed, and twenty tertiary structures were determined. Greater than 70% of the secondary core structures with over 80% alpha helices were correctly identified on protein ranging from 30 to 200 amino-acid sequence. The drift-diffusion model predicted tertiary structures with RMSD values in the 3-5 Angstroms range for proteins ranging 30 to 150 amino acids. These predictions are among the best for an all ab initio protein simulation. Simulations could be run entirely on a desktop computer in minutes; however, more accurate tertiary structures were obtained using molecular dynamic energy relaxation. The drift-diffusion model generated realistic energy versus time traces. Rapid secondary structures followed by a slow compacting towards lower energy tertiary structures occurred after an initial incubation period in agreement with observations.

Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively entangled protein domain structures, it was shown that misfolded entangled subpopulations might become thermosensitive or escape the homeostasis network just after translation. To investigate the role of entanglement in shaping folding kinetics, we introduce a novel indicator and analyze simulations of a coarse-grained, structure-based model for two small single-domain proteins. The model recapitulates the well-known two-state folding mechanism of a non-entangled SH3 domain. However, despite its small size, a natively entangled antifreeze RD1 protein displays a rich refolding behavior, populating two distinct kinetic intermediates: a short-lived, entangled, near-unfolded state and a longer-lived, non-entangled, near-native state. The former directs refolding along a fast pathway, whereas the latter is a kinetic trap, consistently with known experimental evidence of two different characteristic times. Upon trapping, the natively entangled loop folds without being threaded by the N-terminal residues. After trapping, the native entangled structure emerges by either backtracking to the unfolded state or threading through the already formed but not yet entangled loop. Along the fast pathway, trapping does not occur because the native contacts at the closure of the lasso-like loop fold after those involved in the N-terminal thread, confirming previous predictions. Despite this, entanglement may appear already in unfolded configurations. Remarkably, a longer-lived, near-native intermediate, with non-native entanglement properties, recalls what was observed in cotranslational folding.

ColabFold offers accelerated prediction of protein structures and complexes by combining the fast homology search of MMseqs2 with AlphaFold2 or RoseTTAFold. ColabFold's 40-60-fold faster search and optimized model utilization enables prediction of close to 1,000 structures per day on a server with one graphics processing unit. Coupled with Google Colaboratory, ColabFold becomes a free and accessible platform for protein folding. ColabFold is open-source software available at https://github.com/sokrypton/ColabFold and its novel environmental databases are available at https://colabfold.mmseqs.com .

the protein storage vacuole, protein body, and chloroplast. Disulfide transfer pathways that function in the endoplasmic reticulum (ER) and chloroplasts of plants play critical roles in the development of protein storage organelles and the biogenesis of chloroplasts, respectively. Disulfide bond formation requires the cooperative function of disulfide-generating enzymes (e.g., ER oxidoreductase 1), which generate disulfide bonds de novo, and disulfide carrier proteins (e.g., protein disulfide isomerase), which transfer disulfides to substrates by means of thiol-disulfide exchange reactions. Selective molecular communication between disulfide-generating enzymes and disulfide carrier proteins, which reflects the molecular and structural diversity of disulfide carrier proteins, is key to the efficient transfer of disulfides to specific sets of substrates. This review focuses on recent advances in our understanding of the mechanisms and functions of the various disulfide transfer pathways involved in oxidative protein folding in the ER, chloroplasts, and mitochondria of plants.

Molecular chaperones as specialized protein quality control enzymes form the core of cellular protein homeostasis. How chaperones selectively interact with their substrate proteins thus allocate their overall limited capacity remains poorly understood. Here, I present an integrated analysis of sequence and structural determinants that define interactions of protein domains as the basic protein folding unit with the Saccharomyces cerevisiae Hsp70 Ssb. Structural homologs of single-domain proteins that differentially interact with Ssb for de novo folding were found to systematically differ in complexity of their folding landscapes, selective use of nonoptimal codons, and presence of short discriminative sequences, thus highlighting pervasive trade-offs in chaperone-assisted protein folding landscapes. However, short discriminative sequences were found to contribute by far the strongest signal toward explaining Ssb interactions. This observation suggested that some chaperone interactions may be directly programmed in the amino acid sequences rather than responding to folding challenges, possibly for regulatory advantages.

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu-->Glu(-)) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

About 200 mRNA sequences of Escherichia coli and human with matching protein secondary structure data were studied. The mRNA folding for each native sequence and for corresponding randomized sequences was calculated through free energy minimization. We have found that the folding energy of mRNA segments in different protein secondary structures is significantly different. The average Z score is more negative for regular secondary structure (alpha-helix and beta-strand) than that for coil. This suggests that the codon choice in native mRNA sequence coding for protein regular structure contributes more to the mRNA folding stability.

We investigate the rate-length scaling law of protein folding, a key undetermined scaling law in the analytical theory of protein folding. Available data yield statistically significant evidence for the existence of a rate-length law capable of predicting folding times to within about two orders of magnitude (over 9 decades of variation). Unambiguous determination of the functional form of such a law could provide key mechanistic insight into folding. Four proposed laws from literature (power law, exponential, and two stretched exponentials) are tested against one another, and it is found that the power law best explains the data by a modest margin. We conclude that more data is necessary to unequivocally infer the rate-length law. Such data could be obtained through a small number of protein folding experiments on large protein domains.

It is estimated that two-thirds of all proteins in higher organisms are composed of multiple domains, many of them containing discontinuous folds. However, to date, most in vitro protein folding studies have focused on small, single-domain proteins. As a model system for a two-domain discontinuous protein, we study the unfolding/refolding of a slow-folding double mutant of the maltose binding protein (DM-MBP) using single-molecule two- and three-color Förster Resonance Energy Transfer experiments. We observe a dynamic folding intermediate population in the N-terminal domain (NTD), C-terminal domain (CTD), and at the domain interface. The dynamic intermediate fluctuates rapidly between unfolded states and compact states, which have a similar FRET efficiency to the folded conformation. Our data reveals that the delayed folding of the NTD in DM-MBP is imposed by an entropic barrier with subsequent folding of the highly dynamic CTD. Notably, accelerated DM-MBP folding is routed through the same dynamic intermediate within the cavity of the GroEL/ES chaperone system, suggesting that the chaperonin limits the conformational space to overcome the entropic folding barrier. Our study highlights the subtle tuning and co-dependency in the folding of a discontinuous multi-domain protein.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic β-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in β-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases, involving post-translational modifications of the cellular prion protein. Epidemiological studies on Creutzfeldt-Jakob disease, a prototype prion disorder, show a majority of cases being sporadic, while the remaining occurrences are either genetic or iatrogenic. The molecular mechanisms by which PrP(C) is converted into its pathological isoform have not yet been established. While point mutations and seeds trigger the protein to cross the energy barriers, thus causing genetic and infectious transmissible spongiform encephalopathies, respectively, the mechanism responsible for sporadic forms remains unclear. Since prion diseases are protein-misfolding disorders, we investigated prion protein folding and stability as functions of different milieus. Using spectroscopic techniques and atomistic simulations, we dissected the contribution of major structural determinants, also defining the energy landscape of prion protein. In particular, we elucidated (i) the essential role of the octapeptide region in prion protein folding and stability, (ii) the presence of a very enthalpically stable intermediate in prion-susceptible species, and (iii) the role of the disulfide bridge in prion protein folding.

When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enzyme phosphoglycerate kinase (PGK) and of its stable control inside the cell. Unlike the green fluorescent protein control's diffusion coefficient, PGK's diffusion coefficient is a non-monotonic function of temperature, signaling 'sticking' of the protein in the cytosol as it begins to unfold. The temperature-dependent increase and subsequent decrease of the PGK diffusion coefficient in the cytosol is greater than a simple size-scaling model suggests. Chaperone binding of the unfolding protein inside the cell is one plausible candidate for even slower diffusion of PGK, and we test the plausibility of this hypothesis experimentally, although we do not rule out other candidates.