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Staphylokinase Displays Surprisingly Low Mechanical Stability Chengzhi He, and Hongbin Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04425 • Publication Date (Web): 31 Dec 2016 Downloaded from http://pubs.acs.org on January 15, 2017
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Staphylokinase Displays Surprisingly Low Mechanical Stability Chengzhi He# and Hongbin Li* Department of Chemistry University of British Columbia Vancouver, BC V6T 1Z1 Canada
*To whom correspondence should be addressed (
[email protected]). #
Current address: State Key Laboratory of Precision Measurements Technology and
Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072, P. R. China
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Abstract Single molecule force spectroscopy (SMFS) and molecular dynamic (MD) simulations have revealed that the shear topology is an important structural feature for mechanically stable proteins. Proteins that belong to the 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, however, found that staphylokinase (SAK), which shows 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 SAK native state is largely responsible for its low mechanical stability. The molecular origin for this large malleability of SAK remains unknown. Our results reveal a hidden complexity in protein mechanics, and call for detailed investigation into the molecular determinants of the protein mechanical malleability.
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Introduction Protein mechanics plays important roles in a wide variety of biological processes, ranging from cell adhesion1, muscle contraction2,3 to mechanotransduction4,5. Understanding how proteins respond to mechanical stretching force is not only important for elucidating key principles governing various mechanobiological processes, but also important for developing novel protein-based biomaterials with tailored mechanical properties. Thus, understanding how protein mechanical stability is determined by protein structure has been an important research topic in the field of protein mechanics. As an indispensable tool in investigating the nanomechanical properties of macromolecules, ranging from synthetic polymers6-9 to proteins,10-12 atomic force microscope-based single molecule force spectroscopy technique has played important roles in such efforts.10,13-18 AFM studies and molecular dynamics (MD) simulations have synergistically provided invaluable insights into this important question.19-21 Some empirical rules governing the mechanical stability of proteins have started to emerge.10,14-16,19-21 In particular, shear topology of the force-bearing β-strands has been recognized as one key structural feature amongst mechanically stable proteins, where two force-bearing β-strands are organized in parallel. Upon stretching, the two force bearing strands are sheared against each other, and simultaneous rupturing of backbone hydrogen bonds between these two strands constitutes the major barrier to mechanical unfolding of such proteins.20 Thus, this cluster of backbone hydrogen bonds serves as a mechanical clamp to provide major resistance to mechanical unfolding. For example, immunoglobulin-like domains found in the giant muscle protein titin2,3 and proteins of the β-grasp fold, such as ubiquitin,22 GB1,23 protein L24 and SUMO proteins,25 are representative proteins that are mechanically stable and display such mechanical clamps. Moreover, some proteins of such mechanical clamps have been found to unfold at forces as high as 600 pN.26 Developments of MD simulations also made it possible to predict in silico the mechanical stability of proteins whose structures are known.20,21,27-29 In this aspect, Cieplak and colleagues used coarsegrained MD simulations to simulate the mechanical unfolding of a large set of proteins in the Protein Databank (PDB) and predicted their mechanical stability: the survey done in 2007 included 7510 proteins,30 which include all non-fragmented structures with a length between 40 aa and 150 aa deposited in the Protein Data Bank by August 2005, and a later
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survey included 17134 proteins,19 which contains no more than 250 residues and whose structures were deposited in the PDB by December 18, 2008. They observed a good correlation between mechanical unfolding force and protein structure, and identified some putative mechanically stable protein folds.19,30 Consistent with such calculations, proteins that are of significant mechanical stability as revealed in single molecule AFM experiments were also found to be mechanically stable in such in silico calculations. These calculations were very useful in helping identify some extremely mechanically stable proteins, such as cohesin domains.26 Thus, these in silico calculations could provide a helpful means to estimate mechanical stability of proteins that have not been experimentally examined. Despite these progress, it remains unknown if naturally occurring proteins that belong to putative mechanically stable β-grasp fold can be mechanically labile. In an effort to experimentally identify mechanically strong proteins, we used single molecule AFM to characterize the mechanical properties of two structurally highly homologous proteins streptokinase β domain (SKβ)31 and staphylokinase (SAK),32,33 whose structures belong to the putative mechanically stable β-grasp fold, as they are highly ranked in the list of strongest proteins predicted in coarse grained MD simulations.19,30 We found that SKβ is indeed mechanically very stable with an unfolding force of ~220 pN at the pulling speed of 400 nm/s. However, it was very surprising that SAK is mechanically labile and unfolds at ~68 pN under the same condition, making SAK the mechanically weakest protein of the β grasp fold proteins that have been characterized experimentally by using AFM. Our results revealed that the sharp difference in the malleability of the native state of both proteins underscores the dramatic difference in their mechanical unfolding forces. Our results reveal a hidden complexity in protein mechanics, and call for detailed investigation into the molecular determinants of the protein mechanical malleability. Materials and Methods Protein Engineering The plasmids that encode the proteins (SAK and SKβ) were custom synthesized and purchased from GeneScript. The mutant of SAK, SAK11-136, and the mutant of
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SKβ, SKβ-A201W, were constructed using regular polymerase chain reaction and standard site-directed mutagenesis methods, respectively. The genes of polyprotein chimeras (GB1-SKβ)4 , (GB1-SAK)4 and (GB1-SAK11-136)4 were constructed in pQE80L vector using an iterative cloning method that has been previously described (15). Proteins were overexpressed in the DH5α strain induced by isopropyl β-D-1thiogalactopyranoside (IPTG). The Co2+ affinity chromatography was used to purify the proteins. Purified proteins were stored at 4 °C in phosphate buffered saline (PBS) solution at a protein concentration around 1 to 2 mg/mL. Single Molecule Force Spectroscopy Single molecule force spectroscopy experiments were carried out on a custombuilt AFM.34 Before each experiment, the spring constant (which ranged from 30 to 50 pN/nm) of each individual AFM cantilever (MLCT-Bio Si3N4 cantilevers from Bruker) was calibrated using the equipartition theorem35. In a typical AFM experiment, we deposited ∼1 µL of protein solution (1.0 mg/mL) in PBS onto a clean glass coverslip covered by PBS buffer (∼50 µL) and allowed the protein to adsorb onto the substrate for ∼10 min before the force spectroscopy experiment. Monte Carlo Simulations The mechanical unfolding of proteins was described using the Bell-Evans model.36,37 Force-dependent unfolding and refolding rate constants can be described as α(F) = α0exp(FΔxu/kBT), where kB is the Boltzmann constant, T is the absolute temperature in Kelvin, α(F) is the unfolding rate constant at an external force of F, α0 is the unfolding rate constants at zero force, and Δxu is the distance from native to transition states. We carried out Monte Carlo simulations according to previously described procedures1,38 to estimate the kinetic parameters α0 and Δxu, which define the mechanical unfolding of proteins. Equilibrium Denaturation Tryptophan fluoremetry and Circular dichroism (CD) spectroscopy were used for chemical denaturation experiments. Tryptophan fluorescence was measured on a Cary
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Eclipse fluorescence spectrophotometer at 360 nm while the excitation wavelength was 280 nm, and CD measurements were performed on a Jasco-J810 spectropolarimeter in a 0.2-cm path-length cuvette at a scan rate of 50 nm/min. Guanidine hydrochloride (GdmCl) was used as denaturant. The data was fitted by two-state model equation: 𝑌) + 𝛽) 𝑥 + (𝑌. + 𝛽. [𝑥])𝑒 3(45 𝑌𝑜𝑏𝑠 = ∘ 1 + 𝑒 3(45 78[9])/;
