Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery.

Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc-based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat-denatured Nluc dyads and triads can be efficiently refolded by the E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co-chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins.

Ligand-Mediated Mechanical Enhancement in Protein Complexes at Nano- and Macro-Scale.

Protein self-assembly plays a vital role in a myriad of biological functions and in the construction of biomaterials. Although the physical association underlying these assemblies offers high specificity, the advantage often compromises the overall durability of protein complexes. To address this challenge, we propose a novel strategy that reinforces the molecular self-assembly of protein complexes mediated by their ligand. Known for their robust noncovalent interactions with biotin, streptavidin (SAv) tetramers are examined to understand how the ligand influences the mechanical strength of protein complexes at the nanoscale and macroscale, employing atomic force microscopy-based single-molecule force spectroscopy, rheology, and bioerosion analysis. Our study reveals that biotin binding enhances the mechanical strength of individual SAv tetramers at the nanoscale. This enhancement translates into improved shear elasticity and reduced bioerosion rates when SAv tetramers are utilized as cross-linking junctions within hydrogel. This approach, which enhances the mechanical strength of protein-based materials without compromising specificity, is expected to open new avenues for advanced biotechnological applications, including self-assembled, robust biomimetic scaffolds and soft robotics.

Special Issue: 18th Congress of the Polish Biophysical Society.

The 18th Congress of the Polish Biophysical Society took place at the Faculty of Physics of the University of Warsaw in Warsaw, Poland, in September 2022. In total, 111 attendees (Attendance Profile: 107 in-person, 4 remote; Italy 1, Lithuania 1, Poland 104, United Kingdom 1, United States 4) participated in the event. The authors of lectures and posters at the Congress were invited to prepare their presentations in the form of articles in this special issue of the European Biophysics Journal. The 11 articles published in this special issue present a limited sampling of the subjects of the conference presentations. Nevertheless, they showcase excellence in Polish biophysics across a wide range of topics, using both theoretical and experimental approaches: mechanisms of receptor-ligand interactions, medical applications of proteins and nucleic acids, non-linear dynamics/molecular dynamics of protein systems, hydrodynamics and biosensing. We hope to improve on the representation of the international Polish biophysical community after the next Congress in 2025.

Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations.

A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912-Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912 construct. Our SMFS results confirm that Nluc's unfolding proceeds similarly in all constructs; however, Nluc's refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.

Capturing intrinsic nanomechanics of allostery.

The Hsp70 chaperone exploits allosteric communication between its substrate binding domain and its nucleotide binding domain to regulate the loading and release of misfolded polypeptides in an ATP-hydrolysis-dependent manner. In this issue of Biophysical Journal, Singh, Rief, and Zoldák report an exquisitely detailed study of the nanomechanical aspects of the allosteric mechanism in DnaK, an Escherichia coli heat shock protein 70 chaperone.

Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations.

Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies.

Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.

NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

Piecewise All-Atom SMD Simulations Reveal Key Secondary Structures in Luciferase Unfolding Pathway.

Although the folding of single-domain proteins is well characterized theoretically and experimentally, the folding of large multidomain proteins is less well known. Firefly luciferase, a 550 residue three-domain protein, has been commonly used as a substrate to study chaperone reactions and as a model system for the study of folding of long polypeptide chains, including related phenomena such as cotranslational folding. Despite being characterized by various experimental techniques, the atomic-level contributions of various secondary structures of luciferase to its fold's mechanical stability remain unknown. Here, we developed a piecewise approach for all-atom steered molecular dynamics simulations to examine specific secondary structures that resist mechanical unfolding while minimizing the amount of computational resources required by the large water box of standard all-atom steered molecular dynamics simulations. We validated the robustness of this approach with a small NI3C protein and used our approach to elucidate the specific secondary structures that provide the largest contributions to luciferase mechanostability. In doing so, we show that piecewise all-atom steered molecular dynamics simulations can provide novel atomic resolution details regarding mechanostability and can serve as a platform for novel mutagenesis studies as well as a point for comparison with high-resolution force spectroscopy experiments.

Meeting report - NSF-sponsored workshop 'Progress and Prospects of Single-Molecule Force Spectroscopy in Biological and Chemical Sciences'.

The goals of the workshop organized by Piotr Marszalek and Andres Oberhauser that took place between 29 August and 1 September 2019 at Duke University were to bring together leading experts and junior researchers to review past accomplishments, recent advances and limitations in the single-molecule force spectroscopy field, which examines nanomechanical forces in diverse biological processes and pathologies. Talks were organized into four sessions, and two in-depth roundtable discussion sessions were held.

Exploiting a Mechanical Perturbation of a Titin Domain to Identify How Force Field Parameterization Affects Protein Refolding Pathways.

Molecular mechanics force fields have been shown to differ in their predictions of biomolecular processes such as protein folding. To test how force field differences affect predicted polypeptide behavior, we created a mechanically perturbed model of the ß-stranded I91 titin domain based on atomic force spectroscopy data and examined its refolding behavior using six different force fields. We found that different force fields varied significantly in their ability to refold the mechanically perturbed I91 domain. Examination of the perturbed I91 unfolded state revealed that all five Amber force fields oversample a specific region of the Ramachandran plot, thereby creating unfolded state intermediates which are not predicted by the Charmm 22* force field. Simulations of perturbed I91 refolding with Amber FB15 revealed that Amber FB15 destabilizes stable portions of I91, thereby contradicting experimental stability analyses. Finally, inspection of the perturbed I91 unfolded state along with equilibration simulations of the Ac-(AAQAA)3-NH2 peptide suggest that high dihedral torsional barriers cause the Amber ff14SB force field to predict higher helical lifetimes relative to other force fields. These results suggest that using mechanically perturbed models can provide a controlled method to gain insights into how force fields affect polypeptide behavior.

An estimate to the first approximation of microtubule rupture force.

Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.

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.

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.

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.

Endonuclease-independent DNA mismatch repair processes on the lagging strand.

DNA mismatch repair (MMR) pathways coordinate the excision and re-synthesis of newly-replicated DNA if a mismatched base-pair has been identified by protein MutS or MutS homologues (MSHs) after replication. DNA excision during MMR is initiated at single-strand breaks (SSBs) in vitro, and several redundant processes have been observed in reconstituted systems which either require a pre-formed SSB in the DNA or require a mismatch-activated nicking endonuclease to introduce a SSB in order to initiate MMR. However, the conditions under which each of these processes may actually occur in living cells have remained obscured by the limitations of current MMR assays. Here we use a novel assay involving chemically-modified oligonucleotide probes to insert targeted DNA 'mismatches' directly into the genome of living bacteria to interrogate their replication-coupled repair processes quantitatively in a strand-, orientation-, and mismatched nucleotide-specific manner. This 'semi-protected oligonucleotide recombination' (SPORE) assay reveals direct evidence in Escherichia coli of an efficient endonuclease-independent MMR process on the lagging strand-a mechanism that has long-since been considered for lagging-strand repair but never directly shown until now. We find endonuclease-independent MMR is coordinated asymmetrically with respect to the replicating DNA-directed primarily from 3'- of the mismatch-and that repair coordinated from 3'- of the mismatch is in fact the primary mechanism of lagging-strand MMR. While further work is required to explore and identify the molecular requirements for this alternative endonuclease-independent MMR pathway, these findings made possible using the SPORE assay are the first direct report of this long-suspected mechanism in vivo.

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.

Spermidine-Induced Attraction of Like-Charged Surfaces Is Correlated with the pH-Dependent Spermidine Charge: Force Spectroscopy Characterization.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

Warhammers for Peaceful Times.

The Perkins group has recently developed a number of improved atomic force microscopy cantilevers using the focused ion beam technology. They compared the performance of these cantilevers in "real-life" biophysical single-molecule force spectroscopy measurements on protein unfolding, and the results of this comparison are reported in this issue of Biophysical Journal.

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.