Fast Folding Research Articles

How Does a Simplified-Sequence Protein Fold?

To investigate a putatively primordial protein we have simplified the sequence of a 56-residue α/ β fold (the immunoglobulin-binding domain of protein G) by replacing it with polyalanine, polythreonine, and diglycine segments at regions of the sequence that in the folded structure are α-helical, β-strand, and turns, respectively. Remarkably, multiple folding and unfolding events are observed in a 15- μs molecular dynamics simulation at 330 K. The most stable state (populated at ∼20%) of the simplified-sequence variant of protein G has the same α/ β topology as the wild-type but shows the characteristics of a molten globule, i.e., loose contacts among side chains and lack of a specific hydrophobic core. The unfolded state is heterogeneous and includes a variety of α/ β topologies but also fully α-helical and fully β-sheet structures. Transitions within the denatured state are very fast, and the molten-globule state is reached in <1 μs by a framework mechanism of folding with multiple pathways. The native structure of the wild-type is more rigid than the molten-globule conformation of the simplified-sequence variant. The difference in structural stability and the very fast folding of the simplified protein suggest that evolution has enriched the primordial alphabet of amino acids mainly to optimize protein function by stabilization of a unique structure with specific tertiary interactions.

Fast Folding of an RNA Tetraloop on a Rugged Energy Landscape Detected by a Stacking-Sensitive Probe

We investigate the microsecond-timescale kinetics of the RNA hairpin g a ∗ cUUCGguc. The fluorescent nucleotide 2-aminopurine ( a ∗ ) reports mainly on base stacking. Ten kinetic traces and the temperature denaturation curve are globally fitted to four-state models of the free-energy surface. In the best-fitting sequential model, the hairpin unfolds over successively larger barriers in at least three stages: stem fraying and increased base-stacking fluctuations; concerted loss of hydrogen bonding and partial unstacking; and additional unstacking of single strands at the highest temperatures. Parallel and trap models also provide adequate fits: such pathways probably also play a role in the complete free-energy surface of the hairpin. To interpret the model states structurally, 200 ns of molecular dynamics, including six temperature-jump simulations, were run. Although the sampling is by no means comprehensive, five different states were identified using hydrogen bonding and base stacking as reaction coordinates. The four to five states required to explain the experiments or simulations set a lower limit on the complexity of this small RNA hairpin's energy landscape.

NEW LIMITS ON RADIO EMISSION FROM X-RAY DIM ISOLATED NEUTRON STARS

We have carried out a search for radio emission at 820 MHz from six X-ray dim isolated neutron stars (XDINSs) with the Robert C. Byrd Green Bank Radio Telescope. No transient or pulsed emission was found using fast folding, fast Fourier transform, and single-pulse searches. The corresponding flux limits are about 0.01 mJy for pulsed emission, depending on the integration time for the particular source and assuming a duty cycle of 2%, and 20 mJy for single dispersed pulses. These are the most sensitive limits to date on radio emission from XDINSs. There is no evidence for isolated radio pulses, as seen in a class of neutron stars known as rotating radio transients. Our results imply that either the radio luminosities of these objects are lower than those of any known radio pulsars, or they could simply be long-period nearby radio pulsars with high magnetic fields beaming away from the Earth. To test the latter possibility, we would need around 40 similar sources to provide a 1σ probability of at least one of them beaming toward us. We also give a detailed description of our implementation of the Fast Folding Algorithm.

Prediction of the protein folding core: Application to the immunoglobulin fold

We propose an algorithm that allows predicting residues important for the formation of the structure of globular proteins. It relies on a simulation that detects the amino acids presenting a maximum number of neighbours during the early steps of the folding process. They have been called MIR (Most Interacting Residues). Independently, description of the protein structures in fragments with closed ends shows the correlation between these extremities and the core of the globules. These fragments are of rather constant length, typically between 20 and 25 amino acids, and we have previously shown that their extremities are preferentially occupied by MIR. Introduction of rules derived from this fragment analysis of tertiary structures allows to smooth the distribution of MIR, for a better match between TEF ends and MIR. In order to assess this prediction of the folding core, a large family of structures has been used, with sequences as different as possible. A dataset of 56 immunoglobulin structures of various functions but common fold has been used in this study. This fold was chosen because it is one of the most populated with a large amount of data available on its nucleus. In the immunoglobulin domain, “functional and structural load is clearly separated: loops are responsible for binding and recognition while interactions between several residues of the buried core provide stability and fast folding” [1]. We then determined the positions susceptible of high importance for the folding process to occur and compared them to published data, either to High Throw Out Order (HTOO), Conservatism of Conservatism (CoC) or Phi value experiments. It results a reasonable agreement between the positions that we predict and experimental data. Besides, our prediction goes beyond the simple use of a null solvent accessibility of amino acids as a criterion to predict the core. We find the same quality of our prediction on the flavodoxin like superfamily.

Analysis of the Free-Energy Surface of Proteins from Reversible Folding Simulations

Computer generated trajectories can, in principle, reveal the folding pathways of a protein at atomic resolution and possibly suggest general and simple rules for predicting the folded structure of a given sequence. While such reversible folding trajectories can only be determined ab initio using all-atom transferable force-fields for a few small proteins, they can be determined for a large number of proteins using coarse-grained and structure-based force-fields, in which a known folded structure is by construction the absolute energy and free-energy minimum. Here we use a model of the fast folding helical λ-repressor protein to generate trajectories in which native and non-native states are in equilibrium and transitions are accurately sampled. Yet, representation of the free-energy surface, which underlies the thermodynamic and dynamic properties of the protein model, from such a trajectory remains a challenge. Projections over one or a small number of arbitrarily chosen progress variables often hide the most important features of such surfaces. The results unequivocally show that an unprojected representation of the free-energy surface provides important and unbiased information and allows a simple and meaningful description of many-dimensional, heterogeneous trajectories, providing new insight into the possible mechanisms of fast-folding proteins.

Slowing Down Downhill Folding: A Three-Probe Study

The mutant Tyr22Trp/Glu33Tyr/Gly46Ala/Gly48Ala of λ repressor fragment λ6−85 was previously assigned as an incipient downhill folder. We slow down its folding in a cryogenic water-ethylene-glycol solvent (−18 to −28°C). The refolding kinetics are probed by small-angle x-ray scattering, circular dichroism, and fluorescence to measure the radius of gyration, the average secondary structure content, and the native packing around the single tryptophan residue. The main resolved kinetic phase of the mutant is probe independent and faster than the main phase observed for the pseudo-wild-type. Excess helical structure formed early on by the mutant may reduce the formation of turns and prevent the formation of compact misfolded states, speeding up the overall folding process. Extrapolation of our main cryogenic folding phase and previous T-jump measurements to 37°C yields nearly the same refolding rate as extrapolated by Oas and co-workers from NMR line-shape data. Taken together, all the data consistently indicate a folding speed limit of ∼4.5 μs for this fast folder.

Stochastic dynamics of model proteins on a directed graph

A method for reconstructing the potential energy landscape of simple polypeptidic chains is described. We show how to obtain a faithful representation of the energy landscape in terms of a suitable directed graph. Topological and dynamical indicators of the graph are shown to yield an effective estimate of the time scales associated with both folding and equilibration processes. This conclusion is drawn by comparing molecular dynamics simulations at constant temperature with the dynamics on the graph, defined as a temperature-dependent Markov process. The main advantage of the graph representation is that its dynamics can be naturally renormalized by collecting nodes into "hubs" while redefining their connectivity. We show that the dynamical properties at large time scales are preserved by the renormalization procedure. Moreover, we obtain clear indications that the heteropolymers exhibit common topological properties, at variance with the homopolymer, whose peculiar graph structure stems from its spatial homogeneity. In order to distinguish between "fast" and "slow" folders, one has to look at the kinetic properties of the corresponding directed graphs. In particular, we find that the average time needed to the fast folder for reaching its native configuration is two orders of magnitude smaller than its equilibration time while for the bad folder these time scales are comparable.

A Rate-Limiting Conformational Step in the Catalytic Pathway of the glmS Ribozyme

The glmS ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located upstream of the glucosamine-6-phosphate (GlcN6P) synthetase reading frame. Binding of GlcN6P activates site-specific self-cleavage of the glmS mRNA, resulting in the downregulation of glmS gene expression. Unlike other riboswitches, the glmS ribozyme does not undergo structural rearrangement upon metabolite binding, indicating that the metabolite binding pocket is preformed in the absence of ligand. This observation led us to test if individual steps in the reaction pathway could be dissected by initiating the cleavage reaction before or after Mg(2+)-dependent folding. Here we show that self-cleavage reactions initiated with simultaneous addition of Mg(2+) and GlcN6P are slow (3 min(-1)) compared to reactions initiated by addition of GlcN6P to glmS RNA that has been prefolded in Mg(2+)-containing buffer (72 min(-1)). These data indicate that some level of Mg(2+)-dependent folding is rate-limiting for catalysis. Reactions initiated by addition of GlcN6P to the prefolded ribozyme also resulted in a 30-fold increase in the apparent ligand K(d) compared to those of reactions initiated by a global folding step. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. The results of these experiments provided evidence for fast and largely concerted folding of the global tertiary structure (>13 min(-1)). This indicates that the rate-limiting step that we have identified either is a slow folding step between the fast initial folding and ligand binding events or represents the rate of escape from a nativelike folding trap.

Different Members of a Simple Three-Helix Bundle Protein Family Have Very Different Folding Rate Constants and Fold by Different Mechanisms

The 15th, 16th, and 17th repeats of chicken brain α-spectrin (R15, R16, and R17, respectively) are very similar in terms of structure and stability. However, R15 folds and unfolds 3 orders of magnitude faster than R16 and R17. This is unexpected. The rate-limiting transition state for R15 folding is investigated using protein engineering methods (Φ-value analysis) and compared with previously completed analyses of R16 and R17. Characterisation of many mutants suggests that all three proteins have similar complexity in the folding landscape. The early rate-limiting transition states of the three domains are similar in terms of overall structure, but there are significant differences in the patterns of Φ-values. R15 apparently folds via a nucleation–condensation mechanism, which involves concomitant folding and packing of the A- and C-helices, establishing the correct topology. R16 and R17 fold via a more framework-like mechanism, which may impede the search to find the correct packing of the helices, providing a possible explanation for the fast folding of R15.

Arc-Repressor Dimerization on DNA: Folding Rate Enhancement by Colocalization

Multimeric proteins are ubiquitous in many cellular processes that require high levels of regulation. Eukaryotic gene expression is often regulated by a mechanism of combinatorial control that involves the binding of dimeric transcription factors to DNA together with the coordinated activity of additional proteins. In this study, we investigated the dimerization of the Arc-repressor on DNA with the aim of achieving microscopic insight into the possible advantages of interacting with DNA as a complex rather than as a monomeric single-domain protein. We used a computational coarse-grained model in which the protein dynamics was governed by native interactions and protein-DNA interactions were dictated by electrostatic forces. Inspired by previous experimental work that showed an enhanced refolding rate for the Arc-repressor in the presence of DNA and other polyanions, we focused on the mechanism and kinetics of the assembly of Arc monomers in the presence of single- (ssDNA) and double-stranded DNA (dsDNA) molecules in a low-salt concentration environment. The electrostatic interactions that attract the protein to the dsDNA were shown to be fundamental in colocalizing the unfolded Arc chains and in accelerating refolding. Arc monomers bind the dsDNA efficiently and nonspecifically, and search for each other via one-dimensional diffusion. The fastest folding of Arc is observed for DNA of 30 bp. Longer DNA is significantly less efficient in accelerating the Arc refolding rate, since the two subunits search distinct regions of the one-dimensional DNA and are therefore much less colocalized. The probability that the two unfolded chains will meet on 200 bp DNA is similar to that in the bulk. The colocalization of Arc subunits on ssDNA results in much faster folding compared to that obtained on dsDNA of the same length. Differences in the rate of Arc refolding, cooperativity, and the structure of its transition state ensemble introduced by ssDNA and dsDNA molecules demonstrate the important role of colocalization in biological self-assembly processes.

Fast Dynamics of Semiflexible Chain Networks of Self-Assembled Peptides

We present the first neutron spin echo (NSE) measurements of self-assembling peptide hydrogel networks to study the fibril dynamics on the nanometer and nanosecond length and time scales. MAX1 and MAX8 are synthetic beta-hairpin peptides that undergo triggered self-assembly at the nanoscale to form a physically cross-linked network of fibrils with a defined cross-section. When subjected to physiological pH and ionic strength (pH 7.4, 150 mM NaCl), the soluble peptides fold into a beta-hairpin and, subsequently, self-assemble to form a structurally rigid hydrogel stabilized by noncovalent cross-links. The sequence of MAX8 is identical to MAX1 with the exception of one single amino acid substitution that reduces the net charge on the peptide. As a result, faster folding and self-assembly kinetics are observed for MAX8 at the same peptide concentration and identical buffer conditions, and gels with a larger storage modulus are formed. NSE measurements of the peptide hydrogels demonstrate that the self-assembled peptide fibrils can be described as semiflexible chains on nanolength and time scales. Alteration of the peptide sequence affected the nanoscale dynamics of the hydrogels but not to an extent comparable to the large difference observed in the bulk viscoelasticity. Small angle neutron scattering (SANS) of the hydrogels reveals increased scattering for MAX8 at low wavevectors, an indication of a heterogeneous network with a tighter mesh size. Therefore, we conjecture that the difference in elastic modulus arises from differences in assembly kinetics that result in increased fibrillar branching and physical cross-links rather than a change in the fibril nanostructure or persistence length.

The role of disulfide bond formation in the conformational folding kinetics of denatured/reduced lysozyme

Oxidative folding is of vital importance for producing therapeutic proteins in bacteria via recombinant DNA technology since disulfide bonds exist in most pharmaceutical proteins. Although oxidative protein folding has been extensively investigated in vitro, little is explored concerning the role of disulfide formation to protein conformational folding rate. The effects of oxidized (GSSG)/reduced (GSH) glutathione and pH on the conformational folding kinetics of denatured/reduced lysozyme have been studied herein by fluorescence and circular dichroism. It is found that 83% tryptophan residue burial requires disulfide formation, and increasing GSSG concentration greatly accelerates the tertiary structure formation. The fast phase folding rate constant ( k 1) is linearly related to GSSG concentration, indicating the rate-limiting role of mixed-disulfide formation. Moreover, k 1 = 0.006(±0.001) s −1 is likely to be a critical value for judging the determinant of the slow phase folding rate ( k 2), namely, k 2 is controlled by disulfide formation rate only at k 1 < 0.006 s −1. These findings have elucidated the determinants of different folding stages and thus may be beneficial for more efficient control of the oxidative folding of proteins.

Folding kinetics of the human prion protein probed by temperature jump.

Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a beta(t) value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.

Multiple Contact Network Is a Key Determinant to Protein Folding Rates

Understanding the relationship between amino acid sequences and folding rates of proteins is an important task in computational and molecular biology. It has been shown that topological parameters, contact order, long-range order, and total contact distance relate well with protein folding rates. In this work, we have systematically analyzed the influence of amino acid residues that form multiple contacts in protein structures to folding rates of proteins. We observed an inverse relationship between the number of residues with multiple contacts and protein folding rates. Further analysis indicates that multiple contacts are influenced by hydrophobic residues, whereas the role is minimal between the residues that are capable of forming hydrogen bonds. The propensity of multiple contacts forming residues showed that aromatic and hydrophobic residues are dominant in two-state proteins, whereas the polar residues Ser and Thr are also preferred ones in three-state proteins. In addition, multiple contact forming residues are interconnected with each other through contact networks in protein structures. The comparison between slow and fast folding proteins demonstrated the presence of more multiple contact forming residues in slow folding proteins with a limit of 4-6 contacts/residue. These results have been reflected in amino acid sequences in the form of short-, medium-, and long-range contacts, which could discriminate slow and fast folding proteins with an accuracy of 96% using a 5-fold cross-validation method.

A designed protein as experimental model of primordial folding

How do proteins accomplish folding during early evolution? Theoretically the mechanism involves the selective stabilization of the native structure against all other competing compact conformations in a process that involves cumulative changes in the amino acid sequence along geological timescales. Thus, an evolved protein folds into a single structure at physiological temperature, but the conformational competition remains latent. For natural proteins such competition should emerge only near cryogenic temperatures, which places it beyond experimental testing. Here, we introduce a designed monomeric miniprotein (FSD-1ss) that within biological temperatures (330-280 K) switches between simple fast folding and highly complex conformational dynamics in a structurally degenerate compact ensemble. Our findings demonstrate the physical basis for protein folding evolution in a designed protein, which exhibits poorly evolved or primordial folding. Furthermore, these results open the door to the experimental exploration of primitive folding and the switching between alternative protein structures that takes place in evolutionary branching points and prion diseases, as well as the benchmarking of de novo design methods.

Time Resolved Thermodynamics of Fast Protein Folding in Cytochrome c

One of the earliest studies of fast protein folding involved the photolysis of CO bound Cytochorme c in the presence of 4.5 M Gdn-HCl HCl, pH 7.0. These studies revealed fast coordination of non-native His and Met residues resulting in a ‘frustrated’ folding pathway resulting from non-native coordination. These early events occur on timescales of less than 10 μs. Similar fast folding can be initiated at lower Gdn-HCl at pHs above 9.5. Under these conditions the non-native coordination occurs with time constants of ∼ 300 ns and ∼ 3 μs at 22 dg C. Previous time resolved CD spectra suggest that 8% of the native like secondary structure forms in < 1 μs (Goldbeck et al., PNAS (1999), 96, 2782). Here, photoacoustic calorimetry (PAC) has been utilized to probe the thermodynamics associated with fast folding and non-native ligand coordination in CO-Cytochrome c ta pH 12.7 and 350 mM Gdn-HCl. At temperatures below 18 ° C the PAC signals indicate multi-phasic kinetics that can not be fit to simple exponential sums suggesting a distribution of conformations in the presence of CO. The integrated thermodynamics (integrated thermodynamics for processes occurring in < ∼ 20 μs) give an enthalpy change of 5±2 kcal/mol and a molar volume change of −0.5±0.4 mL/mol. Taking into account the enthalpy and volume changes associated with CO photo-release from the heme, an enthalpy change of - 12 kcal/mol and volume change of -6 mL/mol is obtained for the 8% folding as well as non-native His and Met coordination. These results will be discussed within the context of the protein funnel mechanism for fast folding in cytochorme c.

Nitrobenzyl Phototrigger for Fast Protein Folding Processes

Insights into the first steps of protein folding have increased tremendously over the years because of the many experimental and theoretical methods that made it possible to go beyond the millisecond time resolution of standard stopped-flow or rapid mixing techniques. Central to experimental investigations of the fastest steps of protein folding is the development of fast triggering methods. Although various approaches have been used, most of these suffer from significant drawbacks; in particular, the most widely used method of laser-induced temperature jumps generally triggers unfolding.Alternative fast folding triggers make use of photochemical reactions. One promising approach is the photolysis of a nitrobenzyl group, which is based on the observation that incorporation of nitrobenzyl can affect protein structure and protein-protein interaction.1 One main advantage of this method is that photolysis of the non-native nitrobenzyl results in the fully native protein, thus avoiding any potential distortion of the folding reaction by the phototrigger group itself, which cannot be ruled out for most other photochemical folding triggers. However, the time scale of the nitrobenzyl photolysis reaction, and thus the potential of this method for triggering fast folding reactions, had not been determined so far.Recently, a peptide analogue backbone-caged with a photolabile nitrobenzyl group was reported.2 We have used this model for studying the nitrobenzyl-peptide photolysis reaction using nanosecond time-resolved IR spectroscopy. Our experimental results show a time constant of approx. 100 ns for recovery of the uncaged peptide bond, which indicates that nitrobenzyl photolysis is highly suitable for kinetic studies of even the fastest folding processes.[1] S.K. Nandy, R.S. Agnes, D.S. Lawrence, Org. Lett. 2007, 9, 2249.[2] H. Rhee, J. S. Lee, J. Lee, C. Joo, H. Han, M. Cho, J. Phys. Chem. B 2008, 112, 2128.

Ab Initio Determination of Tryptophan Fluorescence Quenching by Histidine Cation in HP35-N27H, Barnase, and T4Lysozyme

The fast folding, 35-residue villin headpiece, HP35, has been at the center of numerous protein folding rate simulations. Eaton et al. have experimentally followed the folding with the N27H mutant, plausibly because the protonated His27 quenches the fluorescence from Trp23 in the folded form by electron transfer, but not when unfolded. Because of this, at least in some simulations of the folding, a major criterion for the folded form is close proximity of His27 to Trp 23. Protonated His is indeed a potent quencher of Trp fluorescence in solution and in some, but not all, proteins. For quenching to occur the energy of the Trp-to-His charge transfer(CT) state must be low enough to be in resonance with the excited state of the Trp. This resonance is dictated by the electric potential difference between the Trp and His due to local protein environment. In some proteins the electric field enables quenching, and in others it does not. We have carried out QM-MM simulations of quenching by His cation for Trp23 in in villin, Trp94 in barnase and Trp138 in Q105H T4 lysozyme using ab initio electronic coupling [Callis et al J. Phys. Chem. B; 2007; 111(35); 10335-10339] Preliminary results indicate that for villin and T4 lysozyme the lowest CT state is that of the amide backbone of Trp. In contrast, the lowest CT state for barnase has His cation as the electron acceptor. We have also computed electronic coupling matrix elements between 3-methylindole and imidazole cation for all three proteins. The average coupling is 58, 329, and 2 cm-1 for the T4 lysozyme, barnase, and villin cases, respectively, suggesting that the change of Trp fluorescence upon folding in villin is not because of queching by His27.

Microsecond Explicit Solvent Molecular Dynamics Simulations of Protein Folding

Explicit solvent molecular dynamics (MD) simulations of protein folding offer information on the protein folding process with tremendous temporal and spatial resolution. Long timescale protein folding trajectories can aid in interpretation of experimentally observed protein folding kinetics, and in the design of new fast-folding mutants. At the same time, protein folding simulations offer a demanding test for MD force fields to assess their accuracy in describing long-timescale conformational transitions in proteins. Until recently, however, little overlap existed between the timescales accessible to simulations and the time required for small proteins to fold. Recent experimental advances have lead to the discovery and characterization of a variety of proteins which fold on the 1-10 microsecond timescale; at the same time, increased computational power has made multiple microsecond timescales accessible for explicit solvent MD simulations.We performed multiple-microsecond folding simulations of two well-characterized fast-folding proteins, namely the villin headpiece subdomain and Pin1 WW domain. The villin headpiece folds to a native state after ∼6 microseconds of molecular dynamics simulation. Furthermore, a common folding mechanism is observed in multiple simulations from different starting conditions, where all secondary structure elements form over 1-2 microseconds after an initial hydrophobic collapse, but the native structure is only obtained after a complete dissociation and rearrangement of the secondary structure elements relative to each other. In the case of the WW domain, the protein misfolds in all simulated folding attempts. Conformational free energy calculations indicate that the WW domain's native state, a three-stranded beta sheet, is significantly higher (∼ 9 kcal/mol) in free energy for the force field (CHARMM22/CMAP) used than several misfolded helical structures obtained from folding simulations. Our results agree with several other recent studies in suggesting a bias toward helical secondary structure in modern MD force fields.

Single Molecule Studies Of Ubiquitin Unfolding

Our group has recently reported the use of a specially designed single molecule nanomixer1 to study the unfolding kinetics of a yellow fluorescent protein (YFP)2 based on the measurements of single-pair fluorescence resonance energy transfer (spFRET), between the intrinsic chromophore and a covalently attached dye, and single-molecule fluorescence two-colour coincidence detection (TCCD). In this work we extend the application of the technology to the small and fast folding ubiquitin doubly labelled for spFRET and TCCD measurements. We firstly explored a variety of labelling strategies and fluorophores for optimal double labelling of ubiquitin, a key issue in application of single molecule fluorescence methods to protein folding/unfolding. We also added a second micropipette to the nanomixer for double-jump experiments to monitor single-molecule refolding. Using this dual labelled sample we have then studied the unfolding and refolding of ubiquitin at the single molecule level.