Unraveling the Mechanical Unfolding Pathways of a Multidomain Protein: Phosphoglycerate Kinase.

Phosphoglycerate kinase (PGK) is a highly conserved enzyme that is crucial for glycolysis. PGK is a monomeric protein composed of two similar domains and has been the focus of many studies for investigating interdomain interactions within the native state and during folding. Previous studies used traditional biophysical methods (such as circular dichroism, tryptophan fluorescence, and NMR) to measure signals over a large ensemble of molecules, which made it difficult to observe transient changes in stability or structure during unfolding and refolding of single molecules. Here, we unfold single molecules of PGK using atomic force spectroscopy and steered molecular dynamic computer simulations to examine the conformational dynamics of PGK during its unfolding process. Our results show that after the initial forced separation of its domains, yeast PGK (yPGK) does not follow a single mechanical unfolding pathway; instead, it stochastically follows two distinct pathways: unfolding from the N-terminal domain or unfolding from the C-terminal domain. The truncated yPGK N-terminal domain unfolds via a transient intermediate, whereas the structurally similar isolated C-terminal domain has no detectable intermediates throughout its mechanical unfolding process. The N-terminal domain in the full-length yPGK displays a strong unfolding intermediate 13% of the time, whereas the truncated domain (yPGKNT) transitions through the intermediate 81% of the time. This effect indicates that the mechanical properties of yPGK cannot be simply deduced from the mechanical properties of its constituents. We also find that Escherichia coli PGK is significantly less mechanically stable as compared to yPGK, contrary to bulk unfolding measurements. Our results support the growing body of observations that the folding behavior of multidomain proteins is difficult to predict based solely on the studies of isolated domains.

Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase.

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarse-grained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding.

Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a ßγ-Crystallin Protein.

Although multidomain proteins predominate the proteome of all organisms and are expected to display complex folding behaviors and significantly greater structural dynamics as compared with single-domain proteins, their conformational heterogeneity and its impact on their interaction with ligands are poorly understood due to a lack of experimental techniques. The multidomain calcium-binding ßγ-crystallin proteins are particularly important because their deterioration and misfolding/aggregation are associated with melanoma tumors and cataracts. Here we investigate the mechanical stability and conformational dynamics of a model calcium-binding ßγ-crystallin protein, Protein S, and elaborate on its interactions with calcium. We ask whether domain interactions and calcium binding affect Protein S folding and potential structural heterogeneity. Our results from single-molecule force spectroscopy show that the N-terminal (but not the C-terminal) domain is in equilibrium with an alternative conformation in the absence of Ca(2+), which is mechanically stable in contrast to other proteins that were observed to sample a molten globule under similar conditions. Mutagenesis experiments and computer simulations reveal that the alternative conformation of the N-terminal domain is caused by structural instability produced by the high charge density of a calcium binding site. We find that this alternative conformation in the N-terminal domain is diminished in the presence of calcium and can also be partially eliminated with a hitherto unrecognized compensatory mechanism that uses the interaction of the C-terminal domain to neutralize the electronegative site. We find that up to 1% of all identified multidomain calcium-binding proteins contain a similarly highly charged site and therefore may exploit a similar compensatory mechanism to prevent structural instability in the absence of ligand.

Chaperones rescue luciferase folding by separating its domains.

Over the last 50 years, significant progress has been made toward understanding how small single-domain proteins fold. However, very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase, 550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (approximately days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force spectroscopy (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains, allowing them to fold in a cotranslational-like sequential process.

Capturing the mechanical unfolding pathway of a large protein with coiled-coil probes.

The folding behaviors and mechanisms of large multidomain proteins have remained largely uncharacterized, primarily because of the lack of appropriate research methods. To address these limitations, novel mechanical folding probes have been developed that are based on antiparallel coiled-coil polypeptides. Such probes can be conveniently inserted at the DNA level, at different positions within the protein of interest where they minimally disturb the host protein structure. During single-molecule force spectroscopy measurements, the forced unfolding of the probe captures the progress of the unfolding front through the host protein structure. This novel approach allows unfolding pathways of large proteins to be directly identified. As an example, this probe was used in a large multidomain protein with ten identical ankyrin repeats, and the unfolding pathway, its direction, and the order of sequential unfolding were unequivocally and precisely determined. This development facilitates the examination of the folding pathways of large proteins, which are predominant in the proteasomes of all organisms, but have thus far eluded study because of the technical limitations encountered when using traditional techniques.

Origin of overstretching transitions in single-stranded nucleic acids.

We combined single-molecule force spectroscopy with nuclear magnetic resonance measurements and molecular mechanics simulations to examine overstretching transitions in single-stranded nucleic acids. In single-stranded DNA and single-stranded RNA there is a low-force transition that involves unwinding of the helical structure, along with base unstacking. We determined that the high-force transition that occurs in polydeoxyadenylic acid single-stranded DNA is caused by the cooperative forced flipping of the dihedral angle formed between four atoms, O5'-C5'-C4'-C3' (γ torsion), in the nucleic acid backbone within the canonical B-type helix. The γ torsion also flips under force in A-type helices, where the helix is shorter and wider as compared to the B-type helix, but this transition is less cooperative than in the B type and does not generate a high-force plateau in the force spectrums of A-type helices. We find that a similar high-force transition can be induced in polyadenylic acid single-stranded RNA by urea, presumably due to disrupting the intramolecular hydrogen bonding in the backbone. We hypothesize that a pronounced high-force transition observed for B-type helices of double stranded DNA also involves a cooperative flip of the γ torsion. These observations suggest new fundamental relationships between the canonical structures of single-and double-stranded DNA and the mechanism of their molecular elasticity.

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

AFM-Based Single-Molecule Force Spectroscopy of Proteins.

Single-molecule force spectroscopy by AFM (AFM-SMFS) is an experimental methodology that allows unequivocal sensitivity and control for investigating and manipulating the mechanical properties of single molecules. The past 20 years of AFM-SMFS has provided numerous breakthroughs in the understanding of the mechanical properties and force-induced structural rearrangements of sugars, DNA, and proteins. Here, we focus on the application of AFM-SMFS to study proteins, since AFM-SMFS has succeeded in providing abundant information about protein folding pathways, kinetics, interactions, and misfolding. In this chapter we describe the experimental procedures for conducting a SMFS-AFM experiment-including purification of protein samples, setup and calibration of the AFM instrumentation, and the thorough and unbiased analysis of resulting AFM data.

Relevance of the Speed and Direction of Pulling in Simple Modular Proteins.

A theoretical framework capable of predicting the first unit that unfolds in pulled modular proteins has been recently introduced, for "fast enough" pulling velocities. Within this picture, we investigate the unfolding pathway in a chain of identical units and predict that the module closest to the pulled end opens first. Steered molecular dynamics of a simple construct, specifically a chain composed of two coiled-coil motives, shows that this is indeed the case. Notwithstanding, the unfolding behavior strongly depends on the terminus (C or N) from which this homopolyprotein is pulled. Therefore, anisotropic features are revealed and seem to play an important role for the observed unfolding pathway.

Direct observation of multimer stabilization in the mechanical unfolding pathway of a protein undergoing oligomerization.

Understanding how protein oligomerization affects the stability of monomers in self-assembled structures is crucial to the development of new protein-based nanomaterials and protein cages for drug delivery. Here, we use single-molecule force spectroscopy (AFM-SMFS), protein engineering, and computer simulations to evaluate how dimerization and tetramerization affects the stability of the monomer of Streptavidin, a model homotetrameric protein. The unfolding force directly relates to the folding stability, and we find that monomer of Streptavidin is mechanically stabilized by 40% upon dimerization, and that it is stabilized an additional 24% upon tetramerization. We also find that biotin binding increases stability by another 50% as compared to the apo-tetrameric form. We used the distribution of unfolding forces to extract properties of the underlying energy landscape and found that the distance to the transition state is decreased and the barrier height is increased upon multimerization. Finally, we investigated the origin of the strengthening by ligand binding. We found that, rather than being strengthened through intramolecular contacts, it is strengthened due to the contacts provided by the biotin-binding loop that crosses the interface between the dimers.

Improving single molecule force spectroscopy through automated real-time data collection and quantification of experimental conditions.

The benefits of single molecule force spectroscopy (SMFS) clearly outweigh the challenges which include small sample sizes, tedious data collection and introduction of human bias during the subjective data selection. These difficulties can be partially eliminated through automation of the experimental data collection process for atomic force microscopy (AFM). Automation can be accomplished using an algorithm that triages usable force-extension recordings quickly with positive and negative selection. We implemented an algorithm based on the windowed fast Fourier transform of force-extension traces that identifies peaks using force-extension regimes to correctly identify usable recordings from proteins composed of repeated domains. This algorithm excels as a real-time diagnostic because it involves <30 ms computational time, has high sensitivity and specificity, and efficiently detects weak unfolding events. We used the statistics provided by the automated procedure to clearly demonstrate the properties of molecular adhesion and how these properties change with differences in the cantilever tip and protein functional groups and protein age.

Single molecule mechanical manipulation for studying biological properties of proteins, DNA, and sugars.

For over 20 years there has been immense biological insight gained using single molecule mechanical measurements to understand properties of biomolecules. This review outlines the field of single molecule mechanics studies and focuses on the manipulation of proteins, DNA, and sugars by single molecule force spectroscopy (SMFS) by atomic force microscopy (AFM). The methods and examples of SMFS by AFM are illustrated using recent advances in protein science including titin elasticity, mechanical unfolding and refolding of α-helical repeat proteins, mechanoenzymatics of thioredoxin and titin kinase, and intermolecular interactions of P-selectin complexes. The possibilities of SMFS to investigate the mechanics of other biopolymers like double- and single-stranded DNA and forced-induced conformational changes in sugars are also discussed. Finally, SMFS and its application to biological processes, like DNA replication, packing and transcription, and DNA methylation are illustrated. These measurements provide a unique and integral part of the development of our knowledge of biochemistry and molecular mechanics.