Protein folding at single-molecule resolution

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Protein folding studied by real-time NMR spectroscopy

Real-time NMR spectroscopy developed to a generally applicable method to follow protein folding reactions. It combines the access to high resolution data with kinetic experiments allowing very detailed insights into the development of the protein structure during different steps of folding. The present review concentrates mainly on the progress of real-time NMR during the last 5 years. Starting from simple 1D experiments, mainly changes of the chemical shifts and line widths of the resonances have been used to analyze the different states populated during the folding reactions. Today, we have a broad spectrum of 1D, 2D, and even 3D NMR methods focusing on different characteristics of the folding polypeptide chains. More than 20 proteins have been investigated so far by these time-resolved experiments and the main results and conclusions are discussed in this report. Real-time NMR provides comprehensive contributions for joining experiment and theory within the 'new view' of protein folding.     

Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements

The kink-turn (k-turn), a new RNA structural motif found in the spliceosome and the ribosome, serves as a specific protein recognition element and as a structural building block. While the structure of the spliceosomal U4 snRNA k-turn/15.5K complex is known from a crystal structure, it is unclear whether the k-turn also exists in this folded conformation in the free U4 snRNA. Thus, we investigated the U4 snRNA k-turn by single-molecule FRET measurements in the absence and presence of the 15.5K protein and its dependence on the Na(+) and Mg(2+) ion concentration. We show that the unfolded U4 snRNA k-turn introduces a kink of 85 degrees +/- 15 degrees in an RNA double helix. While Na(+) and Mg(2+) ions induce this more open conformation of the k-turn, binding of the 15.5K protein was found to induce the tightly kinked conformation in the RNA that increases the kink to 52 degrees +/- 15 degrees . By comparison of the measured FRET distances with a computer-modeled structure, we show that this strong kink is due to the k-turn motif adopting its folded conformation. Thus, in the free U4 snRNA, the k-turn exists only in an unfolded conformation, and its folding is induced by binding of the 15.5K protein.     

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg²⁺ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg²⁺ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg²⁺ concentration that arises from association with several Mg²⁺ ions. This transition is followed by a second Mg²⁺-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ~100ms timescale offers insight into the folding dynamics of this ribozyme.     

Conformational plasticity and dynamics in the generic protein folding catalyst SlyD unraveled by single-molecule FRET

The relation between conformational dynamics and chemistry in enzyme catalysis recently has received increasing attention. While, in the past, the mechanochemical coupling was mainly attributed to molecular motors, nowadays, it seems that this linkage is far more general. Single-molecule fluorescence methods are perfectly suited to directly evidence conformational flexibility and dynamics. By labeling the enzyme SlyD, a member of peptidyl-prolyl cis-trans isomerases of the FK506 binding protein type with an inserted chaperone domain, with donor and acceptor fluorophores for single-molecule fluorescence resonance energy transfer, we directly monitor conformational flexibility and conformational dynamics between the chaperone domain and the FK506 binding protein domain. We find a broad distribution of distances between the labels with two main maxima, which we attribute to an open conformation and to a closed conformation of the enzyme. Correlation analysis demonstrates that the conformations exchange on a rate in the 100 Hz range. With the aid from Monte Carlo simulations, we show that there must be conformational flexibility beyond the two main conformational states. Interestingly, neither the conformational distribution nor the dynamics is significantly altered upon binding of substrates or other known binding partners. Based on these experimental findings, we propose a model where the conformational dynamics is used to search the conformation enabling the chemical step, which also explains the remarkable substrate promiscuity connected with a high efficiency of this class of peptidyl-prolyl cis-trans isomerases.     

Observation of protein folding/unfolding dynamics of ubiquitin trapped in agarose gel by single-molecule FRET

A ubiquitin mutant with two Cys mutations, m[C]q/S65C, was site-specifically labeled with two dye molecules, Alexa Fluor 488 (donor) and Alexa Fluor 594 (acceptor), due to the different reactivity of these two Cys residues. This doubly dye-labeled ubiquitin has lower structural stability than wild-type ubiquitin. Taking advantage of this decreased stability, conformational heterogeneity of this protein under nondenaturing condition was observed at the single-molecule level using single-paired Förster resonance energy transfer (FRET) by trapping the protein in agarose gel. Three conformational populations corresponding to folded (E _ET ≈ 0.95), loosely packed (E _ET ≈ 0.72), and unfolded (E _ET ≈ 0.22) structures, and the structural transitions between them were observed. Our results suggest that agarose immobilization is good for observing structural dynamics of proteins under native condition.     

Determination of the size of lipid rafts studied through single-molecule FRET simulations

Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows the characterization of spatial and temporal properties of biological structures and mechanisms. In this work, we developed an in silico single-molecule FRET methodology to study the dynamics of fluorophores inside lipid rafts. We monitored the fluorescence of a single acceptor molecule in the presence of several donor molecules. By looking at the average fluorescence, we selected events with single acceptor and donor molecules, and we used them to determine the raft size in the range of 5–16 nm. We conclude that our method is robust and insensitive to variations in the diffusion coefficient, donor density, or selected fluorescence threshold.     

A practical guide to single-molecule FRET

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.     

Protein folding mechanisms studied by pulsed oxidative labeling and mass spectrometry

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.     

Analysis of single-molecule FRET trajectories using hidden Markov modeling

The analysis of single-molecule fluorescence resonance energy transfer (FRET) trajectories has become one of significant biophysical interest. In deducing the transition rates between various states of a system for time-binned data, researchers have relied on simple, but often arbitrary methods of extracting rates from FRET trajectories. Although these methods have proven satisfactory in cases of well-separated, low-noise, two- or three-state systems, they become less reliable when applied to a system of greater complexity. We have developed an analysis scheme that casts single-molecule time-binned FRET trajectories as hidden Markov processes, allowing one to determine, based on probability alone, the most likely FRET-value distributions of states and their interconversion rates while simultaneously determining the most likely time sequence of underlying states for each trajectory. Together with a transition density plot and Bayesian information criterion we can also determine the number of different states present in a system in addition to the state-to-state transition probabilities. Here we present the algorithm and test its limitations with various simulated data and previously reported Holliday junction data. The algorithm is then applied to the analysis of the binding and dissociation of three RecA monomers on a DNA construct.     

Protein folding by motion planning

We investigate a novel approach for studying protein folding that has evolved from robotics motion planning techniques called probabilistic roadmap methods (PRMs). Our focus is to study issues related to the folding process, such as the formation of secondary and tertiary structures, assuming we know the native fold. A feature of our PRM-based framework is that the large sets of folding pathways in the roadmaps it produces, in just a few hours on a desktop PC, provide global information about the protein's energy landscape. This is an advantage over other simulation methods such as molecular dynamics or Monte Carlo methods which require more computation and produce only a single trajectory in each run. In our initial studies, we obtained encouraging results for several small proteins. In this paper, we investigate more sophisticated techniques for analyzing the folding pathways in our roadmaps. In addition to more formally revalidating our previous results, we present a case study showing that our technique captures known folding differences between the structurally similar proteins G and L.     

Protein folding assisted by chaperones

Molecular chaperones are one of the most important cell defense mechanisms against protein aggregation and misfolding. These specialized proteins bind non-native states of other proteins and assist them in reaching a correctly folded and functional conformation. Chaperones also participate in protein translocation by membranes, in the stabilization of unstable protein conformers and regulatory factors, in the delivery of substrates for proteolysis and in the recovery of proteins from aggregates.     

Millisecond protein folding studied by NMR spectroscopy

Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecond-to-millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.     

Protein folding

The importance of protein folding in the biosynthesis of proteins is reviewed.     

Heterodimerization of integrin Mac-1 subunits studied by single-molecule imaging

Heterodimerization of integrin Mac-1 (α_Mβ₂) subunits plays important role on regulating leukocytes adhesion to extracellular matrix or endothelial cells. Here, using total internal reflection microscopy, we investigated the heterodimerization of integrin Mac-1 subunits at the single-molecule level in live cells. Individual α_M subunit fused to the enhanced yellow fluorescent protein (eYFP) was imaged at the basal plasma membrane of live Chinese hamster ovary (CHO) cells. Through analysis of mean square displacement (MSD), diffusion coefficient, the size of restricted domain and fraction of molecules undergoing restricted diffusion, we found that as compared with the diffusion in the absence of β₂ subunit, the diffusion of single-molecule of α_M-YFP was suppressed significantly in the presence of β₂ subunit. Thus, based on the oligomerization-induced trapping model, we suggested that in the presence of β₂ subunit, the α_M subunit may form heterodimer with it.