Hongbin Li


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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2021)
Engineering globular protein-based hydrogels with tunable mechanical properties (2021)

Most recently, hydrogels composed of recombinant proteins/peptides have attracted great interest due to their diverse biofunctions, designable amino acids sequence, reactive side chains, excellent biocompatibility and biodegradability. Moreover, conformational switching of folded proteins induces structural and thermodynamic changes in the network, leading to dynamic properties and energy dissipative ability of hydrogels. Despite these advantages, applications of recombinant protein-based hydrogels are often limited by their poor mechanical performance and inefficient network crosslinking, due to steric hindrance of bulky protein domains in the networks. To optimize mechanical behaviors of structural protein-based hydrogels, it is important to understand the design principles of globular protein-based elastomeric networks. This thesis discusses the latest research on protein-based hydrogels, including network designs, crosslinking strategies, and resulting physical and mechanical features of the materials. In addition, classic rubber elasticity theory is introduced to reveal the effects of elastomeric network on physical and mechanical properties of synthetic polymer hydrogels. Based on the theory, a semi-quantitative tool was developed to predict/explain mechanical properties of globular folded polyprotein-based hydrogels, providing guidance for the rational design of globular polyprotein-based hydrogels with desired mechanical properties. Furthermore, we demonstrate for the first time that the macroscopic mechanical energy generated from conformational change of globular proteins closely corresponds to single molecule mechanochemical events by investigating mechanochemical coupling cycles of globular folded proteins at both single-molecule and macroscopic levels.With the guidance of the design principle, we describe synthesis of super tough protein-based hydrogels based on a novel denatured-crosslinking hydrogelation method, which provides potential cartilage-like biomaterials and a general strategy for mechanical enhancement for protein-based hydrogels. We also explored the synthesis of super tough protein-alginate hybrid hydrogels using a double-network approach, further broadening the range of mechanical properties that protein-based biomaterials can reach. Taking advantage of precise mechanical control and dynamic behaviors of mutually exclusive protein-based hydrogels, an application of protein-based hydrogels is reported as extracellular matrices for cell spreading studies, demonstrating a great potential utility of dynamic protein hydrogels in cellular mechano-biology studies.

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Constructing Hydrogels From Engineered Protein (2016)

Hydrogels are crosslinked polymer networks that absorb large amounts of aqueous solutions. The varieties of hydrogel building blocks include natural polymers, synthetic polymers and genetically engineered proteins. This dissertation will discuss the latest progress of hydrogels constructed from genetically engineered proteins (recombinant proteins), mainly focusing on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. Examples include dynamic hydrogels and designing hydrogels via protein fragments reconstitution.The first class of studies presented in this dissertation involves the use of molecular level processes to control macroscopic mechanical properties. A novel protein hydrogel is reported showing dynamic mechanical properties based on a redox potential controlled protein folding-unfolding switch, which is constructed from a designed mutually exclusive protein. The changes of the mechanical and physical properties of this hydrogel are fully reversible and can be applied as extracellular matrix to investigate cell response upon varying stiffness. In addition, another powerful method, metal chelation, is reported to tune the conformational change of mutually exclusive protein, which can stabilize the domain, initiate the folding switch and further affect the mechanical properties of resultant hydrogel.In the second class of studies, protein fragment reconstitution has been demonstrated as a novel driving force for engineering self-assembling reversible protein hydrogels. Protein fragment reconstitution, also known as fragment complementation, is a self-assembling mechanism by which protein fragments can reconstitute the folded conformation of the native protein when split into two halves. GL5 is a small peotein, which is capable of fragment reconstitution spontaneously when split into two halves, GN and GC. Using GL5 as a model, different building blocks are designed to engineer self-assembling, physically crosslinked protein hydrogels. These novel hydrogels show temperature-dependent reversible sol-gel transition, and excellent property against erosion in water. It is anticipated that such fragment reconstitution may offer a general driving force for engineering protein hydrogels from a variety of proteins, expanding the horizon of “bottom-up” approaches in the design principles of biomaterials.

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Force-Induced Topological Changes of Proteins Studied at the Single-Molecule Level (2016)

The knotted polypeptide chain is one of the most surprising topological features found in certain proteins. Understanding how knotted proteins overcome the topological difficulty during the folding process has become a challenging problem. Theoretical studies suggest that a slipknotted structure can serve as an important intermediate in the transformation from unknotted structure to a knotted structure. This thesis mainly focuses on the mechanical folding/unfolding of a slipknotted protein, the ORF109 of the Acidianus Filamentous Virus 3 (AFV3-109), using single-molecule force spectroscopy (SMFS). To show the power of atomic force microscopy (AFM) in the study of SMFS, an α/β protein, NuG2, is stretched and relaxed at a single-molecule level using AFM and the mechanical unfolding and folding events are directly observed. By applying force onto AFV3-109 in different directions, we are able to untie the slipknot to a linear polypeptide chain, as well as tighten it into a trefoil knot involving ∼ 13 amino acid residues. Multiple pathways of untying and tightening are found by both SMFS experiments and Steered Molecular Dynamics (SMD) simulations, revealing that the kinetic partitioning mechanism governs the unfolding of the slipknotted protein. In addition, SMD simulations provide detailed molecular mechanisms of the unfolding of the protein and the topological changes from a slipknot to a linear chain, as well as from a slipknot to a trefoil knot. Moreover, the mechanical folding of AFV3-109 is directly observed using optical tweezers, providing new insight into the folding mechanism of knotted/slipknotted proteins.

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Exploring metal-ligand bonds in metalloproteins by mechanical force (2013)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Tandem modular protein-based hydrogels as extracellular matrix mimetic biomaterials (2013)

New generations of protein-based hydrogels are being developed rapidly over the last several decades for potential applications in biomedicine as well as basic biological studies. Protein-based hydrogels have been explored as synthetic extracellular matrices (ECM) for applications in cell culture and tissue regeneration. A variety of proteins, including silk protein, fibrin, collagen and elastin have been studied. Most of these proteins are non-globular proteins. However, a large number of ECM proteins are tandem modular proteins that consist of many individually folded domains. We hypothesize that tandem modular proteins may also be used in constructing novel hydrogels that can mimic the physical and biochemical characteristics of natural extracellular matrices. In this dissertation, we explored the feasibility of using tandem modular proteins for constructing protein-based biomaterials.First, through self-assembly of two complementary leucine zipper sequences, artificial tandem modular proteins were engineered to form physically cross-linked hydrogels mimicking ECM.Going a step further, a photochemical cross-linking strategy is employed to covalently cross-link engineered artificial elastomeric protein to biomaterials that exhibit mechanical properties mimicking the passive elasticity of muscles. To optimize the biocompatibility of the tandem modular protein-based hydrogels, a protein domain which contains cell-binding sequences is used to construct ECM-mimetic hydrogels. The hydrogels can support cell adhension. Our result also suggests a possible method to design functional hydrogels.To prove the possibility of designing functional hydrogels, an xylanase was used to design enzymatic hydrogels. Our result shows that the enzymes remain active after being cross-linked into hydrogels.The possibility was further proved by fluorescent hydrogels designed from tandem modular protein based on Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP). The fluorescent hydrogels can be applied as force sensors in cells with picoNewton (pN) sensitivity.

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Mechanical Folding/Unfolding of Proteins Probed by Single Molecule Atomic Force Microscopy (2011)

The mechanical folding/unfolding of proteins is involved in many biological processes. However, the molecular mechanism underlining the mechanical folding/unfolding of proteins remains an open question. Most of the current knowledge about the protein folding is from ensemble measurements. In the study of the molecular mechanism underlying the mechanical folding/unfolding of proteins, single molecule atomic force microscopy (AFM) has its unique advantages. Although many endeavors have been made by using single molecule AFM to study the mechanical folding/unfolding of proteins and numerous interesting details have been revealed, the underlying mechanism of protein mechanical folding/unfolding remains largely unknown. The main objective of this thesis is to study the mechanical folding/unfolding of some model proteins using single molecule AFM. First, we studied the mechanical unfolding pathways of two domain-insertion proteins: a natural one, T4-lysozyme (T4L), and an artificially designed one, GL5/T4L (GL5: a mutant of protein GB1). Our study on T4L provided the first direct evidence of the kinetic partitioning assumption for protein folding at the single molecule level. Our study on GL5/T4L revealed its mechanical unfolding pathway with a reversed mechanical unfolding hierarchy. The designing ofdomain-insertion proteins also presented a new concept to program the mechanical unfolding pathway of multi-domain proteins. Second, we studied the mechanical folding/unfolding of TNfn3 domain by combining single molecule AFM with the steered molecular dynamics (SMD) simulation and protein engineering. The mechanical design of TNfn3 was found robust and thebackbone H-bonds of TNfn3 were found critical for its mechanical stability. Our results showed the first direct evidence that the mechanical folding pathways of TNfn3 aregoverned by kinetic partitioning. Third, we studied the folding/unfolding kinetics and mechanics of an artificially designed mutually exclusive protein GL5/I27w34f (I27w34f: a tryptophan removed mutant of I27). The mutually exclusive protein GL5/I27w34f is designed to mimic the natural domain-insertion proteins which are typically difficult to study directly. Our study provided the first direct evidence that protein folding can generate sufficient mechanical strain to unravel a host protein and the folding of mutually exclusive proteins involving a tug-of-war. Mutually exclusive proteins provide a new system for manipulating proteinfolding.

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Engineering proteins of novel mechanical properties : from single molecule to biomaterials (2009)

Elastomeric proteins are an important class of mechanical proteins that take care of the strength, elasticity and extensibility of tissues and biological machineries. Elastomeric proteins are also essential building blocks of materials with outstanding mechanical properties. However, it was largely unknown how elastomeric proteins achieve the remarkable mechanical stability until the advent of single molecule force spectroscopy techniques, such as single molecule atomic force microscopy (AFM). Single molecule AFM has enabled the direct characterization of the mechanical properties of elastomeric proteins at the single molecule level and led to the new promising research area of single protein mechanics and engineering. Combined with molecular dynamics simulation and protein engineering, single molecule AFM has provided rich information about the mechanical design of elastomeric proteins. This dissertation focuses on engineering proteins with novel mechanical properties and makes use of this new technique. It is demonstrated how a non-mechanical protein GB1, the B1 immunoglobulin (IgG) binding domain of protein G from Streptococcus, shows superb mechanical stability. We also investigated the effect of denaturant, guanidium hydrochloride (GdmCl), on the mechanical stability of GB1. It was found that the mechanical stability of GB1 decreases with the increase of GdmCl concentration. Using GB1 as a model system, we demonstrated two ways to enhance the mechanical stability of proteins: by metal chelation and by stabilizing protein-protein interactions. It is revealed that preferentially stabilizing the native state over the mechanical unfolding transition state of proteins is the key to achieve enhanced mechanical stability. We also showed two applications of engineered elastomeric proteins. One is the design of an artificial elastomeric protein with dual mechanical stability that can be regulated reversibly by protein-protein interactions. We introduced proline mutations to GB1 to make it mechanically labile and behave as entropic springs. Upon binding of the Fc fragment of IgG, the proline mutants of GB1 switched into a state of significant mechanical stability and can serve as shock-absorbers. The other application is the engineering of the first tandem modular protein based thermo-reversible hydrogel, which paves the way for engineering hydrogels with much improved physical properties that can be used as artificial extracellular matrices and tissue engineering materials.

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Master's Student Supervision (2010 - 2020)
Mechanical unfolding and folding studies by optical tweezers (2017)

As a single molecule technique, optical tweezers technique proves to be a powerful tool to investigate the physical and chemical properties of DNA/RNA and protein molecules. In this thesis, optical tweezers are applied to two studies. In the first study, we directly investigated the unfolding and folding pathways and kinetics of the wild-type Top7 with optical tweezers. The existence of a folding intermediate state is confirmed. The unfolding process also occasionally shows non-cooperative behavior which has not been observed before. To identify if the mechanical stability of an isolated fragment of Top7 is responsible for the non-cooperative unfolding and folding behavior of Top7, we purified the C-fragment of Top7 and found that it reaches equilibrium at low applied forces, which indicates that Top7’s C-fragment could unfold and fold independently, but the unfolding and folding behavior of Top7 depends on the mutual assistance of both N-terminal and C-terminal residues. Illuminated by computational simulation methods, six residues were mutated aiming at improving the folding cooperativity of Top7. The results show that the folding cooperativity is improved significantly, while the unfolding intermediate appears more frequently. The possible influence of pathways on the frequency of occurrence of unfolding/folding intermediate state is discussed. In the second study, the two-step unfolding behavior of rubredoxin is revealed by optical tweezers. The reversible unfolding/folding behavior under force pressure and chemical pressure are further studied. Optical tweezers technique is proved to be well suited for mechanical unfolding/folding studies of metalloproteins.

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Mechanical unfolding and folding studies on proteins with high sequence identity but different conformations (2015)

Although a few mutations can radically shift the equilibrium between denatured state and native state of a protein, it is surprising that one mutation can switch one fold into another completely different fold. Two Streptococcus binding domains GA and GB could be mutated so that ultimately two completely different folds had only one different amino acid in their sequences. This experiment established a mutational pathway to switch a protein’s fold and function. In order to further understand the mechanism underlying this pathway, single molecule force spectroscopy was carried out using optical tweezers to investigate certain proteins along the mutational pathway to determine their mechanical stability and unfolding/folding kinetics. In this dissertation, GB’s homologous protein NuG2 was studied and demonstrated that the force spectroscopy was a robust and informative tool to determine the unfolding/folding kinetics and the free energy profile of protein unfolding. Additionally, the kinetics and free energy profiles of GA and other mutants including GA30, GA77, GA95 and GB30 were characterized. These results provide a clear tendency of free energy change along the mutational pathway.

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Mechanical denaturation: Forced unfolding of proteins (2013)

Mechanical denaturation has emerged as a novel method to study chemical andphysical properties of protein molecules. In this thesis, single-molecule forcespectroscopy has been carried out using the atomic force microscope to investigatethe mechanical design of proteins through denaturation via an applied mechanicalforce. In the first study, a small globular protein has been shown to exhibitpronounced anisotropic response to directional mechanical stress. One proteincan be both mechanically strong and weak. It will be strong when direction ofthe force vector is aligned with particular structural elements of the protein, andit will be weak otherwise. Mechanical denaturation in the strong direction is accompaniedby cooperative disruption of intramolecular interactions in the protein.Conversely, mechanical denaturation in the weak direction is accompanied by sequentialdisruption of those same interactions. In the second study, the mechanicalproperties of a cofactor dependent protein is characterized. It is shown that boththe protein and cofactor are mechanically strong in the presence of the cofactor.Removal of the cofactor tremendously diminishes the mechanical strength of theprotein. The mutually supportive roles of structure and function are demonstratedthrough mechanical denaturation experiments.

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Investigating Mechanical Properties of a Protein by Single Molecule Atomic Force Microscopy (2012)

Single molecule AFM is a powerful technique affording the opportunity to understand the mechanical properties of proteins at the level of a single molecule. In Combining with the protein engineering techniques, single molecule allows us to understand protein folding/unfolding mechanisms and develop methods to tune the mechanical stability. Here, we use a small protein, GB1, the B1 IgG binding domain of protein G from Streptococcus, as a model system. In this thesis, we employed bi-His metal binding sites to probe the mechanical unfolding transition state of GB1 and rationally enhance the mechanical stability of GB1 mutant, G6-53. The transition state cannot be trapped and detected by the usual structural methods because of its high free energy. It remains a challenging task and research focus. In Chapter 3, we directly probed the mechanical unfolding transition state structure of protein GB1. The results demonstrate that the contacts between the force-bearing strands 1 and 4 are largely disrupted at the transition state, whereas the first β-hairpin and α-helix were largely intact. The second hairpin was partially disrupted. These results are in close agreement with, and provide a benchmark for, MD simulations.The mechanical stability is critical for the overall mechanical properties of elastomeric proteins. Elastomeric proteins provide tissues with extensibility, elasticity, and mechanical strength. In Chapter 4, we enhanced the mechanical stability of G6-53 with different metal ions. We demonstrated that all four divalent metal ions, Ni²⁺, Co²⁺, Zn²⁺ and Cu²⁺, enhance the mechanical stability of G6-53 to different degrees. Because this process is completely reversible, the protein can be treated like a switch. Moreover, the resultant unfolding force difference between Co²⁺ and Zn²⁺ or Zn²⁺ and Cu²⁺ is ~ 20 pN. Thus, various metal ions can be used to fine tune the mechanical stability of proteins.

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