Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox.

Single-molecule force spectroscopy sheds light onto the free energy landscapes governing protein folding and molecular recognition. Since only a single molecule or single molecular complex is probed at any given point in time, the technique is capable of identifying low-probability conformations within a large ensemble of possibilities. It furthermore allows choosing certain unbinding pathways through careful selection of the points at which the force acts on the protein or molecular complex. This review focuses on recent innovations in construct design, site-specific bioconjugation, measurement techniques, instrumental advances, and data analysis methods for improving workflow, throughput, and data yield of AFM-based single-molecule force spectroscopy experiments. Current trends that we highlight include customized fingerprint domains, peptide tags for site-specific covalent surface attachment, and polyproteins that are formed through mechanostable receptor-ligand interactions. Recent methods to improve measurement stability, signal-to-noise ratio, and force precision are presented, and theoretical considerations, analysis methods, and algorithms for analyzing large numbers of force-extension curves are further discussed. The various innovations identified here will serve as a starting point to researchers in the field looking for opportunities to push the limits of the technique further.

Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods.

During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data.

Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level.

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.

The mechanical hierarchies of fibronectin observed with single-molecule AFM.

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.

Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of ß-Barrel Assembly Machinery Polypeptide Transport Associated Domains.

Single-molecule force spectroscopy by atomic force microscopy exploits the use of multimeric protein constructs, namely, polyproteins, to decrease the impact of nonspecific interactions, to improve data accumulation, and to allow the accommodation of benchmarking reference domains within the construct. However, methods to generate such constructs are either time- and labor-intensive or lack control over the length or the domain sequence of the obtained construct. Here, we describe an approach that addresses both of these shortcomings that uses Gibson assembly (GA) to generate a defined recombinant polyprotein rapidly using linker sequences. To demonstrate the feasibility of this approach, we used GA to make a polyprotein composed of alternating domains of I27 and TmCsp, (I27-TmCsp)3-I27)(GA), and showed the mechanical fingerprint, mechanical strength, and pulling speed dependence are the same as an analogous polyprotein constructed using the classical approach. After this benchmarking, we exploited this approach to facilitiate the mechanical characterization of POTRA domain 2 of BamA from E. coli (EcPOTRA2) by assembling the polyprotein (I27-EcPOTRA2)3-I27(GA). We show that, as predicted from the α + ß topology, EcPOTRA2 domains are mechanically robust over a wide range of pulling speeds. Furthermore, we identify a clear correlation between mechanical robustness and brittleness for a range of other α + ß proteins that contain the structural feature of proximal terminal ß-strands in parallel geometry. We thus demonstrate that the GA approach is a powerful tool, as it circumvents the usual time- and labor-intensive polyprotein production process and allows for rapid production of new constructs for single-molecule studies. As shown for EcPOTRA2, this approach allows the exploration of the mechanical properties of a greater number of proteins and their variants. This improves our understanding of the relationship between structure and mechanical strength, increasing our ability to design proteins with tailored mechanical properties.

Biophysical investigations of engineered polyproteins: implications for force data.

Dynamic force spectroscopy is rapidly becoming a standard biophysical technique. Significant advances in the methods of analysis of force data have resulted in ever more complex systems being studied. The use of cloning systems to produce homologous tandem repeats rather than the use of endogenous multidomain proteins has facilitated these developments. What is poorly addressed are the physical properties of these constructed polyproteins. Are the properties of the individual domains in the construct independent of one another or attenuated by adjacent domains? We present data for a construct of eight fibronectin type III domains from the human form of tenascin that exhibits approximately 1 kcal mol(-1) increase in stability compared to the monomer. This effect is salt and pH dependent, suggesting that the stabilization results from electrostatic interactions, possibly involving charged residues at the interfaces of the domains. Kinetic analysis shows that this stabilization reflects a slower unfolding rate. Clearly, if domain-domain interactions affect the unfolding force, this will have implications for the comparison of absolute forces between types of domains. Mutants of the tenascin 8-mer construct exhibit the same change in stability as that observed for the corresponding mutation in the monomer. And when Phi-values are calculated for the 8-mer construct, the pattern is similar to that observed for the monomer. Therefore, mutational analyses to resolve mechanical unfolding pathways appear valid. Importantly, we show that interactions between the domains may be masked by changes in experimental conditions.

Single protein misfolding events captured by atomic force microscopy.

Using single protein atomic force microscopy (AFM) techniques we demonstrate that after repeated mechanical extension/relaxation cycles, tandem modular proteins can misfold into a structure formed by two neighboring modules. The misfolding is fully reversible and alters the mechanical topology of the modules while it is about as stable as the original fold. Our results show that modular proteins can assume a novel misfolded state and demonstrate that AFM is able to capture, in real time, rare misfolding events at the level of a single protein.