Single-Molecule Force-Clamp Spectroscopy: Dwell Time Analysis and Practical Considerations

Single-molecule force-clamp spectroscopy has become a powerful tool for studying protein folding/unfolding, bond rupture, and enzymatic reactions. Different methods have been developed to analyze force-clamp spectroscopy data on polyproteins to obtain kinetic parameters characterizing the mechanical unfolding of proteins, which are often modeled as a two-state process (a Poisson process). However, because of the finite number of domains in polyproteins, the statistical analysis of the force-clamp spectroscopy data is different from that of a classical Poisson process, and the equivalency of different analysis methods remains to be proven. In this article, we show that these methods are equivalent and lead to accurate measurements of the unfolding rate constant. We also demonstrate that distinct from the constant-pulling-velocity experiments, in which the unfolding rate extracted from the data is dependent on the number of protein domains in the polyproteins (the N effect), force-clamp experiments do not show any N effect. Using a simulated data set, we also highlighted important practical considerations that one needs to take into account when using the single-molecule force-clamp spectroscopy technique to characterize the unfolding energy landscape of proteins

A Facile Way to Tune Mechanical Properties of Artificial Elastomeric Proteins-Based Hydrogels

Protein-based hydrogels have attracted considerable interests due to their potential applications in biomedical engineering and material sciences. Using a tandem modular protein (GB1)8 as building blocks, we have engineered chemically cross-linked hydrogels via a photochemical cross-linking strategy, which is based on the cross-linking of two adjacent tyrosine residues into dityrosine adducts. However, because of the relatively low reactivity of tyrosine residues in GB1, (GB1)8-based hydrogels exhibit poor mechanical properties. Here, we report a Bolton–Hunter reagent-based, facile method to improve and tune the mechanical properties of such protein-based hydrogels. Using Bolton–Hunter reagent, we can derivatize lysine residues with phenolic functional groups to modulate the phenolic (tyrosine-like) content of (GB1)8. We show that hydrogels made from derivatized (GB1)8 with increased phenolic content show significantly improved mechanical properties, including improved Young’s modulus, breaking modulus as well as reduced swelling. These results demonstrate the great potential of this derivatization method in constructing protein-based biomaterials with desired macroscopic mechanical properties

Two-Molecule Force Spectroscopy on Proteins

Many elastomeric proteins, which play important roles in a wide range of biological processes, exist as parallel/antiparallelly arranged dimers or multimers to perform their mechanobiological functions. For example, in striated muscle sarcomeres, the giant muscle protein titin exists as hexameric bundles to mediate the passive elasticity of muscles. However, it has not been possible to directly probe the mechanical properties of such parallelly arranged elastomeric proteins. And it remains unknown if the knowledge obtained from single-molecule force spectroscopy studies can be directly extrapolated to such parallelly/antiparallelly arranged systems. Here, we report the development of atomic force microscopy (AFM)-based two-molecule force spectroscopy to directly probe the mechanical properties of two elastomeric proteins that are arranged in parallel. We developed a twin-molecule approach to allow two parallelly arranged elastomeric proteins to be picked up and stretched simultaneously in an AFM experiment. Our results clearly revealed the mechanical features of such parallelly arranged elastomeric proteins during force–extension measurements and allowed for the determination of mechanical unfolding forces of proteins in such an experimental setting. Our study provides a general and robust experimental strategy to closely mimic the physiological condition of such parallel elastomeric protein multimers

Domain Insertion Effectively Regulates the Mechanical Unfolding Hierarchy of Elastomeric Proteins: Toward Engineering Multifunctional Elastomeric Proteins

The architecture of elastomeric proteins controls fine-tuned nanomechanical properties of this class of proteins. Most elastomeric proteins are tandem modular in structure, consisting of many individually folded domains of varying stability. Upon stretching, these elements unfold sequentially following a strict hierarchical pattern determined by their mechanical stability, where the weakest element unfolds first and the strongest unfolds last. Although such a hierarchical architecture is well-suited for biological functions of elastomeric proteins, it may become incompatible with incorporating proteins of desirable functionality in order to construct multifunctional artificial elastomeric proteins, as many of these desired proteins are not evolved for mechanical purpose. Thus, exposure to a high stretching force will result in unraveling of these proteins and lead to a loss of their functionality. To overcome this challenge, we combine protein engineering with single molecule force spectroscopy to demonstrate that domain insertion is an effective strategy to control the mechanical unfolding hierarchy of multidomain proteins and effectively protect mechanically labile domains. As a proof-of-principle experiment, we spliced a mechanically labile T4 lysozyme (T4L) into a flexible loop of a mechanically stronger host domain GL5 to create a domain insertion protein. Using single molecule force spectroscopy, we showed that the mechanically labile T4L domain unfolds only after the mechanically stronger host domain GL5 has unfolded. Such a reverse mechanical unfolding hierarchy effectively protects the mechanically labile T4L domain from applied stretching force and significantly increased the lifetime of T4L. The approach demonstrated here opens the possibility to incorporate labile proteins into elastomeric proteins for engineering novel multifunctional elastomeric proteins

Direct Observation of Tug-of-War during the Folding of a Mutually Exclusive Protein

Although most protein folding studies are carried out on single-domain proteins, over two-thirds of proteins in proteomes are built up from multiple individually folded domains. A significant fraction of these multidomain proteins are domain-insertion proteins, in which one guest domain is inserted into a surface loop of a host protein. Intricate thermodynamic and kinetic coupling between the two domains can have a profound impact on their folding dynamics. Here we use an engineered mutually exclusive protein as a model system to directly illustrate one such complex dynamic process: the “tug-of-war” process during protein folding. By inserting a guest protein I27w34f into a host protein GB1-L5 (GL5), we engineered a novel, mutually exclusive protein, GL5/I27w34f, in which only one domain can remain folded at any given time due to topological constraints imposed by the folded structures. Using stopped-flow techniques, we obtained the first kinetic evidence that the guest and host domains engage in a folding tug-of-war as they attempt to fold, in which the host domain folds rapidly into its three-dimensional structure and is then automatically unfolded, driven by the folding of the guest domain. Our results provided direct evidence that protein folding can generate sufficient mechanical strain to unravel a host protein. Using single-molecule atomic force microscopy, we provide direct evidence for the existence of a conformational equilibrium between the two mutually exclusive conformations. Our results highlight important roles played by the intricate coupling between folding kinetics, thermodynamic stability, and mechanical strain in the folding of complex multidomain proteins, which cannot be addressed in traditional single-domain protein folding studies

Highly Covalent Ferric−Thiolate Bonds Exhibit Surprisingly Low Mechanical Stability

Depending on their nature, different chemical bonds show vastly different stability with covalent bonds being the most stable ones that rupture at forces above nanonewton. Studies have revealed that ferric−thiolate bonds are highly covalent and are conceived to be of high mechanical stability. Here, we used single molecule force spectroscopy techniques to directly determine the mechanical strength of such highly covalent ferric−thiolate bonds in rubredoxin. We observed that the ferric−thiolate bond ruptures at surprisingly low forces of ∼200 pN, significantly lower than that of typical covalent bonds, such as C−Si, S−S, and Au−thiolate bonds, which typically ruptures at >1.5 nN. And the mechanical strength of Fe−thiolate bonds is observed to correlate with the covalency of the bonds. Our results indicated that highly covalent Fe−thiolate bonds are mechanically labile and display features that clearly distinguish themselves from typical covalent bonds. Our study not only opens new avenues to investigating this important class of chemical bonds, but may also shed new lights on our understanding of the chemical nature of these metal thiolate bonds

Staphylokinase Displays Surprisingly Low Mechanical Stability

Single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations have revealed that shear topology is an important structural feature for mechanically stable proteins. Proteins containing a β-grasp fold display the typical shear topology and are generally of significant mechanical stability. In an effort to experimentally identify mechanically strong proteins using single-molecule atomic force microscopy, we found that staphylokinase (SAK), which has a typical β-grasp fold and was predicted to be mechanically stable in coarse-grained MD simulations, displays surprisingly low mechanical stability. At a pulling speed of 400 nm/s, SAK unfolds at ∼60 pN, making it the mechanically weakest protein among the β-grasp fold proteins that have been characterized experimentally. In contrast, its structural homologous protein streptokinase β domain displays significant mechanical stability under the same experimental condition. Our results showed that the large malleability of native-state SAK is largely responsible for its low mechanical stability. The molecular origin of this large malleability of SAK remains unknown. Our results reveal a hidden complexity in protein mechanics and call for a detailed investigation into the molecular determinants of the protein mechanical malleability

Protein Hydrogels with Reversibly Patterned Multidimensional Fluorescent Images for Information Storage

Fluorescent polymeric hydrogels are promising soft and wet media for information storage that are desirable for lifelike biomaterials and flexible electronics. Hydrogels based on engineered proteins have attracted considerable interest. However, their potential utility as information storage media has remained largely unexplored. Here, we report a protein-based hydrogel that can serve as an information storage medium. Using LOVTRAP, which consists of protein LOV2 and its binding partner ZDark1, we developed a novel strategy to decorate/release fluorescent proteins onto/from a blank protein hydrogel slate in light-controlled and spatially defined fashions, reversibly generating fluorescent patterns such as quick response codes. To increase the information storage capacity, we further developed grayscale patterning to generate pseudo-colored multi-dimensional fluorescent images. Results of this new method demonstrate a novel reversible information storage approach in soft and wet materials and open a new avenue toward developing next-generation protein-based smart materials for information storage and anti-counterfeit applications