Folding Time Scales Research Articles

Helix Formation from Hydrogen Bond Kinetics in Alanine Homopeptides

We present an analysis of α-helix folding in the coarse-grained coordinate of number of formed helical hydrogen bonds (NHBs) for four alanine peptides (ALA)n, with n = 5, 8, 15, and 21 residues. Starting with multi-microsecond all-atom molecular dynamics trajectories in aqueous solution, we represent the system dynamics in a space of between four (for ALA5) and twenty (for ALA21) hydrogen-bonding microstates. In all cases, transitions changing the hydrogen bond count by 1–2 dominate and the coil formation, NHB 1 → 0, is the fastest process. The calculation of global maximum weight paths shows that, when analyzed at a sufficiently long lag time, folding in the NHB coordinate is consecutive, with direct folding, 0 → 3, for ALA5 and bottlenecks at transitions 4 → 6 for ALA8, 0 → 5 for ALA15, and 0 → 9 for ALA21. Further coarse-graining to 2–4 dimensions was performed with the optimal dimensionality reduction method, allowing the identification of crucial folding intermediates and time scales of their formation in ALA8, ALA15, and ALA21. The detailed analysis of hydrogen bonding patterns revealed that folding is initiated preferentially at both peptide termini. The kinetic model was also used to estimate diffusion and friction coefficients for helix propagation. The description of the helix formation process in the hydrogen bonding coordinate NHB was in good general agreement with the experimental data and qualitatively similar to previous kinetic models of higher dimensions based on structural clustering. Use of the low-dimensional hydrogen bonding picture thus provides a different, complementary way of describing the complex and fascinating mechanism of helix formation.

Molecular Latent Space Simulators for Distributed and Multimolecular Trajectories.

All atom molecular dynamics (MD) simulations offer a powerful tool for molecular modeling, but the short time steps required for numerical stability of the integrator place many interesting molecular events out of reach of unbiased simulations. The popular and powerful Markov state modeling (MSM) approach can extend these time scales by stitching together multiple short discontinuous trajectories into a single long-time kinetic model but necessitates a configurational coarse-graining of the phase space that entails a loss of spatial and temporal resolution and an exponential increase in complexity for multimolecular systems. Latent space simulators (LSS) present an alternative formalism that employs a dynamical, as opposed to configurational, coarse graining comprising three back-to-back learning problems to (i) identify the molecular system's slowest dynamical processes, (ii) propagate the microscopic system dynamics within this slow subspace, and (iii) generatively reconstruct the trajectory of the system within the molecular phase space. A trained LSS model can generate temporally and spatially continuous synthetic molecular trajectories at orders of magnitude lower cost than MD to improve sampling of rare transition events and metastable states to reduce statistical uncertainties in thermodynamic and kinetic observables. In this work, we extend the LSS formalism to short discontinuous training trajectories generated by distributed computing and to multimolecular systems without incurring exponential scaling in computational cost. First, we develop a distributed LSS model over thousands of short simulations of a 264-residue proteolysis-targeting chimera (PROTAC) complex to generate ultralong continuous trajectories that identify metastable states and collective variables to inform PROTAC therapeutic design and optimization. Second, we develop a multimolecular LSS architecture to generate physically realistic ultralong trajectories of DNA oligomers that can undergo both duplex hybridization and hairpin folding. These trajectories retain thermodynamic and kinetic characteristics of the training data while providing increased precision of folding populations and time scales across simulation temperature and ion concentration.

TPP Riboswitch Populates Holo-Form-like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration.

Riboswitches are noncoding RNA that regulate gene expression by folding into specific three-dimensional structures (holo-form) upon binding by their cognate ligand in the presence of Mg2+. Riboswitch functioning is also hypothesized to be under kinetic control requiring large cognate ligand concentrations. We ask the question under thermodynamic conditions, can the riboswitches populate structures similar to the holo-form only in the presence of Mg2+ and absence of cognate ligand binding. We addressed this question using thiamine pyrophosphate (TPP) riboswitch as a model system and computer simulations using a coarse-grained model for RNA. The folding free energy surface (FES) shows that with the initial increase in Mg2+ concentration ([Mg2+]), the aptamer domain (AD) of TPP riboswitch undergoes a barrierless collapse in its dimensions. On further increase in [Mg2+], intermediates separated by barriers appear on the FES, and one of the intermediates has a TPP ligand-binding competent structure. We show that site-specific binding of the Mg2+ aids in the formation of tertiary contacts. For [Mg2+] greater than physiological concentration, AD folds into a structure similar to the crystal structure of the TPP holo-form even in the absence of the TPP ligand. The folding kinetics shows that TPP AD populates an intermediate due to the misalignment of two arms present in the structure, which acts as a kinetic trap, leading to larger folding timescales. The predictions of the intermediate structures from the simulations are amenable for experimental verification.

Slow Folding of a Helical Protein: Large Barriers, Strong Internal Friction, or a Shallow, Bumpy Landscape?

The rate at which a protein molecule folds is determined by opposing energetic and entropic contributions to the free energy that shape the folding landscape. Delineating the extent to which they impact the diffusional barrier-crossing events, including the magnitude of internal friction and barrier height, has largely been a challenging task. In this work, we extract the underlying thermodynamic and dynamic contributions to the folding rate of an unusually slow-folding helical DNA-binding domain, PurR, which shares the characteristics of ultrafast downhill-folding proteins but nonetheless appears to exhibit an apparent two-state equilibrium. We combine equilibrium spectroscopy, temperature-viscosity-dependent kinetics, statistical mechanical modeling, and coarse-grained simulations to show that the conformational behavior of PurR is highly heterogeneous characterized by a large spread in melting temperatures, marginal thermodynamic barriers, and populated partially structured states. PurR appears to be at the threshold of disorder arising from frustrated electrostatics and weak packing that in turn slows down folding due to a shallow, bumpy landscape and not due to large thermodynamic barriers or strong internal friction. Our work highlights how a strong temperature dependence on the pre-exponential could signal a shallow landscape and not necessarily a slow-folding diffusion coefficient, thus determining the folding timescales of even millisecond folding proteins and hints at possible structural origins for the shallow landscape.

Kinetic Mechanism of RNA Helix-Terminal Basepairing—A Kinetic Minima Network Analysis

RNA functions are often kinetically controlled. The folding kinetics of RNAs involves global structural changes and local nucleotide movement, such as base flipping. The most elementary step in RNA folding is the closing and opening of a basepair. By integrating molecular dynamics simulation, master equation, and kinetic Monte Carlo simulation, we investigate the kinetics mechanism of RNA helix-terminal basepairing. The study reveals a six-state folding scheme with three dominant folding pathways of tens, hundreds, and thousands of nanoseconds of folding timescales, respectively. The overall kinetics is rate limited by the detrapping of a misfolded state with the overall folding time of 10−5 s. Moreover, the analysis examines the different roles of the various driving forces, such as the basepairing and stacking interactions and the ion binding/dissociation effects on structural changes. The results may provide useful insights for developing a basepair opening/closing rate model and further kinetics models of large RNAs.

Detailed Kinetics of RNA Folding Pathways and Thermodynamic Origins of Crowding by Single-Molecule FRET

Specific three dimensional folding is required for RNA function while RNA can be a highly dynamic system that actively samples different configurations under thermal fluctuations. For instance, in gene regulation of RNA riboswitch, the prompt structural transition in response to ligand is crucial for triggering transcriptional termination, making the time scale of folding and ligand response exceedingly relevant to RNA functions. In single-molecule FRET (smFRET) experiments, the conformational changes of RNA are visualized by the time-dependent energy transfer to determine folding and unfolding rates. Such kinetic information allows us to construct a model for manganese riboswitch folding and explore the detailed folding mechanisms, where Mg2+ binding is found to pre-fold the RNA, followed by ligand (Mn2+) association to further stabilize the folded structure. Moreover, the thermodynamic origins can be obtained from temperature dependent folding. The manganese riboswitch folding is found unexpectedly promoted by heat and the process is endothermic with net entropy gain, much contradictory to the conventional idea of folding into a more order state. In molecular crowding study where we try to understand the influence of fundamental excluded volume on RNA structures in highly concentrated intracellular environments, the chemically inert crowders are shown to entropically favor the more compact folded structure of RNA; a simple physical picture of osmotic pressure is employed to explain the crowder size dependence. Lastly, a hydraulic pressure generator is used to apply pressure (up to 5 kbar) to RNA sample through capillary. The free volume change (pΔV) during RNA folding may be related to structural response under crowding conditions.

Evidence for short ∼ 1 Myr lifetimes from the He ii proximity zones of z ∼ 4 quasars

The duration of quasar accretion episodes is a key quantity for distinguishing between models for the formation and growth of supermassive black holes, the evolution of quasars, and their potential feedback effects on their host galaxies. However, this critical timescale, often referred to as the quasar lifetime, is still uncertain by orders of magnitude ($t_{\rm Q} \simeq 0.01\,{\rm Myr} - 1\,{\rm Gyr}$). Absorption spectra of quasars exhibiting transmission in the He II Ly$\alpha$ forest provide a unique opportunity to make precise measurements of the quasar lifetime. Indeed, the size of a quasar's He II proximity zone, the region near the quasar itself where its own radiation dramatically alters the ionization state of the surrounding intergalactic medium (IGM), depends sensitively on its lifetime for $t_{\rm Q}\lesssim 30$ Myr, comparable to the expected $e$-folding time-scale for SMBH growth $t_{\rm S}=45$ Myr. In this study we compare the sizes of He II proximity zones in the Hubble Space Telescope (HST) spectra of 6 z~4 quasars to theoretical models generated by post-processing cosmological hydrodynamical simulations with $1$D radiative transfer algorithm. We introduce a Bayesian statistical method to infer the lifetimes of individual quasars which allows us to fully marginalize over the unknown ionization state of the surrounding IGM. We measure lifetimes $0.63^{+0.82}_{-0.40}$ Myr and $5.75^{+4.72}_{-2.74}$ Myr for two objects. For the other 4 quasars large redshift uncertainties undermine our sensitivity allowing us to only place upper or lower limits. However a joint analysis of these four systems yields a measurement of their average lifetime of $\langle t_{\rm Q}\rangle = 1.17^{+1.77}_{-0.84}$ Myr. We discuss our short ~1 Myr inferred lifetimes in the context of other quasar lifetime constraints and the growth of SMBHs.

Criteria for folding in structure-based models of proteins.

In structure-based models of proteins, one often assumes that folding is accomplished when all contacts are established. This assumption may frequently lead to a conceptual problem that folding takes place in a temperature region of very low thermodynamic stability, especially when the contact map used is too sparse. We consider six different structure-based models and show that allowing for a small, but model-dependent, percentage of the native contacts not being established boosts the folding temperature substantially while affecting the time scales of folding only in a minor way. We also compare other properties of the six models. We show that the choice of the description of the backbone stiffness has a substantial effect on the values of characteristic temperatures that relate both to equilibrium and kinetic properties. Models without any backbone stiffness (like the self-organized polymer) are found to perform similar to those with the stiffness, including in the studies of stretching.

Monitoring Backbone Hydrogen-Bond Formation in β-Barrel Membrane Protein Folding.

β-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire β-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale.

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

Abstractβ‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.

Mapping fast protein folding with multiple-site fluorescent probes

Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

Proteome folding kinetics is limited by protein halflife.

How heterogeneous are proteome folding timescales and what physical principles, if any, dictate its limits? We answer this by predicting copy number weighted folding speed distribution – using the native topology – for E.coli and Yeast proteome. E.coli and Yeast proteomes yield very similar distributions with average folding times of 100 milliseconds and 170 milliseconds, respectively. The topology-based folding time distribution is well described by a diffusion-drift mutation model on a flat-fitness landscape in free energy barrier between two boundaries: i) the lowest barrier height determined by the upper limit of folding speed and ii) the highest barrier height governed by the lower speed limit of folding. While the fastest time scale of the distribution is near the experimentally measured speed limit of 1 microsecond (typical of barrier-less folders), we find the slowest folding time to be around seconds (8 seconds for Yeast distribution), approximately an order of magnitude less than the fastest halflife (approximately 2 minutes) in the Yeast proteome. This separation of timescale implies even the fastest degrading protein will have moderately high (96%) probability of folding before degradation. The overall agreement with the flat-fitness landscape model further hints that proteome folding times did not undergo additional major selection pressures – to make proteins fold faster – other than the primary requirement to “sufficiently beat the clock” against its lifetime. Direct comparison between the predicted folding time and experimentally measured halflife further shows 99% of the proteome have a folding time less than their corresponding lifetime. These two findings together suggest that proteome folding kinetics may be bounded by protein halflife.

Kinetics study on the HIV-1 ectodomain protein quaternary structure formation reveals coupling of chain folding and self-assembly in the refolding cascade.

Entry of HIV-1 into the target cell is mediated by the envelope glycoprotein consisting of noncovalently associated surface subunit gp120 and transmembrane subunit gp41. To form a functional gp41 complex, the protein undergoes hairpin formation and self-assembly. The fusion event can be inhibited by gp41-derived peptides at nanomolar concentration and is highly dependent on the time of addition, implying a role of folding kinetics on the inhibitory action. Oligomerization of the gp41 ectodomain was demonstrated by light scattering measurements. Kinetic study by stopped-flow fluorescence and absorption measurements (i) revealed a multistate folding pathway and stable intermediates; (ii) showed a dissection of fast and slow components for early and late stages of folding, respectively, with 3 orders of magnitude difference in the time scale; (iii) showed the slow process was attributed to misfolding and unzipping of the hairpin; and (iv) showed retardation of the native hairpin formation is assumed to lead to coupling of the correctly registered hairpin and self-assembly. This coupling allows the deduction on the time scale of intrachain folding (0.1-1 s) for the protein. The folding reaction was illustrated by a free energy profile to explain the temporal dichotomy of fast and slow steps of folding as well as effective inhibition by gp41-derived peptide.

Electrostatically Accelerated Encounter and Folding for Facile Recognition of Intrinsically Disordered Proteins

Achieving facile specific recognition is essential for intrinsically disordered proteins (IDPs) that are involved in cellular signaling and regulation. Consideration of the physical time scales of protein folding and diffusion-limited protein-protein encounter has suggested that the frequent requirement of protein folding for specific IDP recognition could lead to kinetic bottlenecks. How IDPs overcome such potential kinetic bottlenecks to viably function in signaling and regulation in general is poorly understood. Our recent computational and experimental study of cell-cycle regulator p27 (Ganguly et al., J. Mol. Biol. (2012)) demonstrated that long-range electrostatic forces exerted on enriched charges of IDPs could accelerate protein-protein encounter via “electrostatic steering” and at the same time promote “folding-competent” encounter topologies to enhance the efficiency of IDP folding upon encounter. Here, we further investigated the coupled binding and folding mechanisms and the roles of electrostatic forces in the formation of three IDP complexes with more complex folded topologies. The surface electrostatic potentials of these complexes lack prominent features like those observed for the p27/Cdk2/cyclin A complex to directly suggest the ability of electrostatic forces to facilitate folding upon encounter. Nonetheless, similar electrostatically accelerated encounter and folding mechanisms were consistently predicted for all three complexes using topology-based coarse-grained simulations. Together with our previous analysis of charge distributions in known IDP complexes, our results support a prevalent role of electrostatic interactions in promoting efficient coupled binding and folding for facile specific recognition. These results also suggest that there is likely a co-evolution of IDP folded topology, charge characteristics, and coupled binding and folding mechanisms, driven at least partially by the need to achieve fast association kinetics for cellular signaling and regulation.

A “slow” protein folds quickly in the end

Until just a few years ago, it was not clear whether it would be possible to fold proteins using all-atom molecular dynamics simulations with explicit solvent molecules, despite the insights that these simulations had yielded into other biological problems. This was not just because of the computational challenge of reaching folding time scales, typically microseconds to seconds or longer, but also because it was not generally accepted that the empirical energy functions (“force fields”) used were sufficiently accurate to locate the folded state as a global free-energy minimum. This has changed dramatically in the last 2 y, with the development by Shaw and coworkers of a special-purpose supercomputer, Anton, capable of running biomolecular MD simulations on a microsecond or even millisecond timescale (1). This group showed that, with only minor adjustments to existing force fields (2⇓–4), it was possible to fold 12 small, “fast-folding” proteins (1), which adopt their native structure in microseconds. At the time, it was still not clear, however, whether it would be possible to do the same for larger, slower-folding proteins (5, 6). In PNAS, Piana et al. report simulations of ubiquitin folding, which occurs in milliseconds (7). Their results not only extend the computationally accessible time scale for calculating equilibrium folding trajectories by 2–3 orders of magnitude but have a number of implications that can only be deduced from a comparison of fast and slow folding proteins.

Fluctuations and differential contraction during regeneration of Hydra vulgaris tissue toroids**This paper is dedicated to Malcolm Steinberg.

We studied regenerating bilayered tissue toroids dissected from Hydra vulgaris polyps and relate our macroscopic observations to the dynamics of force-generating mesoscopic cytoskeletal structures. Tissue fragments undergo a specific toroid–spheroid folding process leading to complete regeneration towards a new organism. The time scale of folding is too fast for biochemical signalling or morphogenetic gradients, which forced us to assume purely mechanical self-organization. The initial pattern selection dynamics was studied by embedding toroids into hydro-gels, allowing us to observe the deformation modes over longer periods of time. We found increasing mechanical fluctuations which break the toroidal symmetry, and discuss the evolution of their power spectra for various gel stiffnesses. Our observations are related to single-cell studies which explain the mechanical feasibility of the folding process. In addition, we observed switching of cells from a tissue bound to a migrating state after folding failure as well as in tissue injury. We found a supra-cellular actin ring assembled along the toroid's inner edge. Its contraction can lead to the observed folding dynamics as we could confirm by finite element simulations. This actin ring in the inner cell layer is assembled by myosin-driven length fluctuations of supra-cellular F-actin bundles (myonemes) in the outer cell layer.

In Vivo Translation Rates Can Substantially Delay the Co-Translational Folding of the E. Coli Cytosolic Proteome

A question of fundamental importance concerning protein folding in vivo is whether the kinetics of translation or the thermodynamics of the ribosome nascent chain (RNC) complex is the major determinant of co-translational folding behavior. This is because the rate of translation can reduce the probability of co-translational folding below that associated with arrested ribosomes, whose behavior is determined by the equilibrium thermodynamics of the RNC complex. Here, we combine a chemical kinetic equation with genomic and proteomic data to predict domain folding probabilities as a function of nascent chain length for cytosolic proteins of E. coli synthesized on both arrested and continuously translating ribosomes. Our results indicate that, at the translation rates found in vivo, about one-third of the cytosolic proteins of E. coli exhibit co-translational folding, with at least one domain in each of these proteins folding into its stable native structure before the full-length protein is released from the ribosome. The majority of these co-translational folding domains are influenced by translation kinetics which reduces their probability of co-translational folding and consequently increases the nascent chain length at which they fold into their native structures. For about 20% of all cytosolic proteins this delay in folding can exceed the length of the completely synthesized protein, causing one or more of their domains to switch from co- to post-translational folding solely as a result of the in vivo translation rates. Large, β-strand rich domains are more likely to be affected by translation kinetics during co-translational folding than small, α-helical domains that generally fold much faster. These kinetic effects arise from the difference in time scales of folding and amino-acid addition, and they represent a source of metastability for the E. coli proteome.

In vivo translation rates can substantially delay the cotranslational folding of the Escherichia coli cytosolic proteome

A question of fundamental importance concerning protein folding in vivo is whether the kinetics of translation or the thermodynamics of the ribosome nascent chain (RNC) complex is the major determinant of cotranslational folding behavior. This is because translation rates can reduce the probability of cotranslational folding below that associated with arrested ribosomes, whose behavior is determined by the equilibrium thermodynamics of the RNC complex. Here, we combine a chemical kinetic equation with genomic and proteomic data to predict domain folding probabilities as a function of nascent chain length for Escherichia coli cytosolic proteins synthesized on both arrested and continuously translating ribosomes. Our results indicate that, at in vivo translation rates, about one-third of the Escherichia coli cytosolic proteins exhibit cotranslational folding, with at least one domain in each of these proteins folding into its stable native structure before the full-length protein is released from the ribosome. The majority of these cotranslational folding domains are influenced by translation kinetics which reduces their probability of cotranslational folding and consequently increases the nascent chain length at which they fold into their native structures. For about 20% of all cytosolic proteins this delay in folding can exceed the length of the completely synthesized protein, causing one or more of their domains to switch from co- to posttranslational folding solely as a result of the in vivo translation rates. These kinetic effects arise from the difference in time scales of folding and amino-acid addition, and they represent a source of metastability in Escherichia coli's proteome.

Hydrophobic forces and the length limit of foldable protein domains

To find the native conformation (fold), proteins sample a subspace that is typically hundreds of orders of magnitude smaller than their full conformational space. Whether such fast folding is intrinsic or the result of natural selection, and what is the longest foldable protein, are open questions. Here, we derive the average conformational degeneracy of a lattice polypeptide chain in water and quantitatively show that the constraints associated with hydrophobic forces are themselves sufficient to reduce the effective conformational space to a size compatible with the folding of proteins up to approximately 200 amino acids long within a biologically reasonable amount of time. This size range is in general agreement with the experimental protein domain length distribution obtained from approximately 1,200 proteins. Molecular dynamics simulations of the Trp-cage protein confirm this picture on the free energy landscape. Our analytical and computational results are consistent with a model in which the length and time scales of protein folding, as well as the modular nature of large proteins, are dictated primarily by inherent physical forces, whereas natural selection determines the native state.

A simple model predicts experimental folding rates and a hub-like topology.

A simple model is presented that describes general features of protein folding, in good agreement with experimental results and detailed all-atom simulations. Starting from microscopic physics, and with no free parameters, this model predicts that protein folding occurs remarkably quickly because native-like states are kinetic hubs. A hub-like network arises naturally out of microscopic physical concerns, specifically the kinetic longevity of native contacts during a search of globular conformations. The model predicts folding times scaling as τ(f) ~ e(ξN) in the number of residues, but because the model shows ξ is small, the folding times are much faster than Levinthal's approximation. Importantly, the folding time scale is found to be small due to the topology and structure of the network. We show explicitly how our model agrees with generic experimental features of the folding process, including the scaling of τ(f) with N, two-state thermodynamics, a sharp peak in C(V), and native-state fluctuations.