Browsing by Author "Marszalek, Piotr E"

This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.

Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties, and force-induced conformational changes, (2) AFM imaging that permits us to identify topology of single molecules and supramolecular structures, and (3) protein engineering that allows us to genetically connect elastomeric proteins and self-assembling linkers together to construct well-defined protein building blocks.

Nanospring behavior of á-helical repeat proteins: We revealed that á-helical repeat proteins, composed of tightly packed á-helical repeats that form spiral-shaped protein structures, unfold and refold in near equilibrium, while they are stretched and relaxed during AFM based SMFS measurements. In addition to minimal energy dissipation by the equilibrium process, we also found that these proteins can yield high stretch ratios (>10 times) due to their packed initial forms. Therefore, we, for the first time, recognized a new class of polypeptides with nanospring behaviors.

Protein-based force probes for gauging molecular interactions: We developed protein-based force probes for simple, robust and general AFM assays to accurately measure intermolecular forces between self-oligomerization of two or more arbitrary polypeptides that potentially can serve as molecular crosslinkers. For demonstration, we genetically connected the force probe to the Strep-tag II and mixed it with its molecular self-assembling partner, the Strep-Tactin. Clearly characterized force fingerprints by the force probe allowed identification of molecular interactions of the single Strep-tag II and Strep-Tactin complex when the complex is stretched by AFM. We found a single energy barrier exists between Strep-tag II and Strep-Tactin in our given loading rates. Based upon our demonstration, the use of the force probe can be expanded to investigate the strength of interactions within many protein complexes composed of homo- and hetero-dimers, and even higher oligomeric forms. Obtained information can be used to choose potential self-assembling crosslinkers which can connect elastomeric proteins with appropriate strength in higher-order structures.

Self-assembled nano-structured biomaterials with well-defined protein-based building blocks: We constructed well-defined protein building blocks with tailored mechanical properties for self-assembled nano-structured materials. We engineered protein constructs composed of tandem repeats of either a I27-SNase dimer or a I27 domain alone and terminated them with a monomeric streptavidin which is known to form extremely stable tetramers naturally. By using molecular biology and AFM imaging techniques, we found that these protein building blocks transformed into stable tetrameric complexes. By using AFM based SMFS, we measured, to our knowledge for the first time, the mechanical strength of the streptavidin tetramer at a single-molecule level and captured its mechanical anisotropy. Using streptavidin tetramers as crosslinkers offers a unique opportunity to create well-defined protein based self-assembled materials that preserve the molecular properties of their building blocks.

Proteins are molecules found in all living kingdoms due to their several cellular functions. They are comprised of amino acids, which are linked together forming long polypeptide chains. These chains can fold into specific three dimensional structures, unique to every protein, which are directly related to their function. However, proteins can lose this structure for various reasons (e.g., increase of temperature). This can result in protein misfolding and possibly formation of aggregates that are dangerous to cell viability. Some of these aggregates are known to result in diseases, such as Alzheimer's disease.Additionally, in cells there is a class of proteins, the molecular chaperones. Those have been shown to assist other proteins to fold into their native state, along with assist in breaking down aggregates to prevent cell death. However, the mechanisms in which chaperones are able to do so, are not fully understood and are still under investigation. Up till now, only a small number of proteins have been used in the chaperones mechanism studies, despite the large number of substrate proteins they assist. Therefore, it is likely that our current view of the chaperones mechanism may be limited with regards to their overall mechanism. At the same time, with the recent developments in the biotechnology industry, understanding and predicting functional protein folding structures has had a great impact in the development of therapeutic proteins. Here, we aim to examine both the protein unfolding and refolding of a newly identified protein, NanoLuc (Nluc). Nluc is a highly bioluminescent protein, despite its small 19 kDa size. In comparison, Nluc is approximately 3 times smaller than firefly luciferase (Fluc, 61 kDa), a protein that has been extensively used in molecular chaperone assisted refolding studies. However, Nluc produces 150 times brighter bioluminescence signal to Fluc. Additionally, Nluc bioluminescence reaction is ATP independent, unlike Fluc who requires ATP energy to produce the bioluminescence signal. This bioluminescence signal, regardless of the protein, is directly related to the protein’s native state. Thus, any structural compromise in Nluc due to denaturing conditions will result in loss of its bioluminescence signal in bioluminescence assays. Combining this with chaperone proteins, we aim to examine how chaperones assist the refolding of thermally denatured Nluc. In this way we aim to introduce various substrates of Nluc repeats with the goal of identifying new possible chaperone and substrate interactions. Additionally, we engineered novel poly-protein constructs with tandem repeats of Nluc and titin I91 domains and performed mechanical unfolding and misfolding experiments. The aim behind these experiments was to examine the misfolding and refolding behavior of Nluc when it is linked to itself versus when it is separated by the titin I91 domains. Our findings showed that the unfolding behavior of the various constructs were very similar to one another, however their refolding was different. We observed via cyclic mechanical unfolding/refolding studies that when Nluc proteins were separated with titin I91 domains, the protein was less likely to misfold, than when the Nlucs were linked next to each other. A more detailed description per chapter is provided below. In chapter one, we introduce basic principles of biochemistry and physics to be later used. Those include defining what is a protein, introducing thermodynamic laws, discussing the protein folding paradox and the energy landscapes of proteins, and finishing with an introduction to molecular chaperones. In chapter two, we introduce the materials and methods used in this work. We start with explaining what bioluminescence is. Then, we proceed with introducing Nluc along with the engineered protein constructs used in this work. We describe our detailed protocols on protein expression and purification. A description of our bioluminescence protocol is also provided. Then we proceed with an introduction to the Atomic Force Microscopy (AFM)-based Single Molecule Force Spectroscopy (SMFS) technique, which relies on the elastic properties of the polypeptide chain of proteins. Worm-like chain model is explained along with basic operation of the device used during the experiments. Lastly, we introduce Molecular Dynamic (MD) Simulations, and we present the protocol followed in those simulations. In chapter three, we present the AFM-based SMFS experiments for one of the Nluc protein constructs, ?912−????3−?912. In this chapter we examine and discuss the unfolding behavior of this poly-Nluc and titin I91 domain construct in which the three Nlucs are linked next to each other by a very short linker (5 amino acids). The aim is to understand how linking the Nluc repeats in close proximity affects their misfolding behavior. In chapter four, similarly to chapter three, we present the AFM-based SMFS experiments for two of the engineered Nluc protein constructs, ?912−????−?914 and ?91−????−?91−????−?91−????−?91. In one of these constructs, we have the same number of Nluc proteins (three) and titin I91 domains (four), as the construct in chapter three, but in this construct the Nlucs are separated from each other by titin I91 domains. By mechanically unfolding/refolding this construct, we can examine how separating the various Nlucs affects their unfolding and refolding behavior. We also examined the unfolding and refolding behavior of a single Nluc flanked by titin I91 domains. The two constructs in this chapter were compared in order to examine if the three Nlucs behaved as three “single” Nlucs, like in the ?912−????−?914 construct. Our findings showed they had similar behavior. Lastly, we present results on the simulations for steered MD simulations along with coarse-grained MD simulations. The coarse-grained MD simulations were performed by Dr. Pan Zhang, a PhD candidate at that time in Dr. Weitao Yang’s group as part of a collaboration. In chapter five, we tested three protein constructs we engineered that we used in our chaperone assisted refolding studies via bioluminescence assays. The constructs generated were monomeric, dyad, and triad Nluc, which we thermally denatured at the melting temperature of 58?? in the absence of chaperones. During our experiments we observed that monomeric Nluc demonstrated strong thermal stability (minimal loss in bioluminescence signal), as previously reported in literature. However, dyad and triad Nluc constructs would lose their bioluminescence signal indicating a loss in their native three-dimensional conformation. Therefore, we performed chaperone assisted refolding studies to examine the recovery of these proteins, and we observed both were strong chaperone substrates. Another interesting observation was the fact that GrpE, a known nucleotide exchange factor of DnaK, did not have a significant effect in refolding of these constructs. This is contrary to the literature up till now as in most cases GrpE is shown to be very important in the refolding of substrate proteins, e.g., in Fluc refolding assays, as we observed in our control experiments. Additionally, we performed MD simulations to try and elucidate possible misfolding of the various Nluc constructs. We performed both coarse-grained simulations (by our collaborator Dr. Pan Zhang and Dr. Weitao Yang) and all-atoms simulations. Lastly, we present the TADOSS domain swapping simulations results from our collaborators (Dr. Pan Zhang and Dr. Weitao Yang) to provide some computational understanding to possible misfolding events of poly-Nluc constructs. In chapter six, we conclude our findings and present future work on our study.

Large multi-domain proteins, which are ubiquitous in the proteomes in eukaryotic and prokaryotic organisms, still lack intensive studies on their folding mechanisms due to their complicated interactions between and inside of their domains. My work is broadly aimed at characterize folding behaviors and mechanisms of large, multi-domain proteins.

In the first part of my thesis work, I developed a novel mechanical folding polypeptide probe based on the anti-parallel coiled-coil domain of a natural protein (Archeal Box C/D sRNP Core Protein) to enable us to capture the progress of the unfolding front along the host protein structure. In this way, the structural origin of the signal from single molecule force spectroscopy (SMFS) based on atomic force microscopy (AFM) can be directly identified. Beyond this published work, I also characterized and compared the unfolding pathway of two homologous multi-domain proteins – yeast phosphoglycerate kinase (PGK) and its E.coli homolog. I found that yeast PGK has much higher mechanical stability than E.coli PGK although previous literature reported that they had similar thermodynamics stability determined by bulk measurements. In collaboration with Mr. Zackary N. Scholl, we characterized another multi-domain protein, protein S, and its two isolated domains (protein S N terminal domain and C terminal domain). By matching the statistical distributions of the unfolding forces from the truncated domains with the distributions of forces from full protein S, we solved the problem of assigning force peaks in the force-extension (FE) curves to individual domains. However, accurate determination of the structural correspondence to the force peaks still needs insertion of CC probes into the loop regions of these multi-domain proteins.

In the second part of my thesis work, I focused on integrating our AFM-SMFS with fluorescence microscopy methods. In particular, I have focused on constructing and mounting a home-made AFM force spectrometer on a high magnification inverted microscope to monitor fluorescence signal changes when stretching a single protein molecule. I am also setting up a total internal reflection fluorescence microscope (TIRF) for combination of Förster resonance energy transfer (FRET) and SMFS measurements in the future.

In Chapter 1, I will discuss background of multi-protein unfolding, SMFS method principles and coarse-grained (CG) steered molecular dynamics (SMD) simulation. This chapter will focus on the importance of understanding multi-protein unfolding pathway, the advantage of using SMFS as an experimental way of characterizing multi-domain protein unfolding behavior. As a theoretical way to analyze what event happened during the forced unfolding of multi-domain protein, CG-SMD simulations usually support our SMFS results, and providing details for interpreting FE curves obtained in SMFS measurements.

In Chapter 2, I present my recently completed work on developing a mechanical force probe based on an anti-parallel coiled-coil polypeptide chain. This work has just been published by Angewandte Chemie International Edition. The probe I developed provides a new way to determine the structural relation of the peaks shown in protein unfolding FE curves.

In Chapter 3, I report my recently started work on characterization of yeast PGK. I will show the preliminary data we obtained, (this work is also in collaboration with Mr. Zackary N. Scholl), and discuss differences in the FE traces of the two homologs and propose future work directions.

In Chapter 4, I briefly mention another work, Ca2+dependence of protein S unfolding pathway. The NPS and CPS domains were also individually studied by AFM, and their unfolding data statistics helped exploring comprehensively the structural origin of full protein S unfolding data.

In Chapter 5, I will introduce my completed work on combination of AFM and inverted Zeiss microscope. The completion of this work will enable simultaneous recording of the fluorescence signal in an SMFS experiment performed on a single molecule in the future.

In Chapter 6, I will describe my recent work on building a TIRF microscope to realize TPM measurement on single molecule multi-domain protein in our lab. We hope that the integrated instrument could enable us to detect FRET signal coming from unzipping of the CC probe when stretching by AFM.

Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.

Nucleic acids are subjected to many different mechanical loadings inside. These loadings could cause large deformations and conformational changes to these molecules. This is why the mechanical properties of nucleic acids are so important to their functions. Here we use a newly designed and built high-performance AFM force spectrometer, supplemented with molecular dynamics simulations and NMR spectroscopy to investigate the relationship between mechanical properties and structure of different nucleic acids.

To test the mechanical properties of nucleic acids, we successfully designed and purpose-built a single molecule puller, an instrument to physically stretch single molecules, at a fraction of the cost of a commercial AFM instrument. This instrument has similar force noise to hybrid instruments, while also exhibiting significantly lower drift, on the order of five times lower. This instrument allows the measurement of subtle transitions as a molecule is stretched. With the addition of a lock-in amplifier, we possibly could obtain better force resolution, the order of femtonewtons.

We find that helical structure does indeed have an effect on the mechanical properties of double-stranded DNA. As the A-form double helix has a shorter, wider structure compared to the B-form helix, its force spectra exhibit a shorter initial length before the overstretching force plateau, compared to B-form DNA. Contrarily, the Z-form double helix has a narrower, more extended helical structure than B-form DNA, and we see this fact manifest in the force spectra of Z-DNA, which has a longer initial length before the overstretching force plateau. Also, interestingly, we find that neither A, nor Z-DNA force spectra display the second melting force plateau. Indicating this plateau is not necessarily cause by melting of strands apart, but rather a feature of B-DNA.

To better understand the forces that stabilized these different structures, specifically base stacking, we also mechanically characterize different single-stranded helical polynucleotides using AFM based force spectroscopy. We expand on previous studies by confirming that single helical polynucleotides undergo a force transition at a force of ~20 pN as they are uncoiled, and also demonstrating, that when stretched beyond this force transition, the molecules behave differently depending on base sequence and backbone sugar. Specifically, the force spectra of poly-adenylic acid possess a linear force region, which persists to ~300 pN, after the force plateau. We also observe that poly-deoxyadenylic acid is comparatively stiffer than other polynucleotides after undergoing two force transitions. By supplementing our force spectroscopic data with MD simulations and NMR spectroscopy, we find that base stacking in adenine is quite strong, persisting above 100 pN. We find that initial helical structure, which is defined by base stacking and backbone sugar, guides the stretching pathway of the polynucleotides. This finding can possibly be extrapolated to the elasticity of double-stranded DNA.

Soft biomaterials are used in a variety of applications such as scaffolds for cell growth and coatings for implants or transplants. We aim to create a protein hydrogel that will self-assemble upon the mixing of two different protein constructs. This is accomplished using Streptavidin, a protein that tetramerizes, and SpyTag-SpyCatcher, a protein-peptide that spontaneously forms covalent bonds, as the crosslinking mechanisms. Further, using protein building blocks whose viscoelastic properties are known from single-molecule force spectroscopy (SMFS), we aim to create a hydrogel whose physical properties are tailorable by altering the building blocks incorporated in the constructs. This thesis focuses on using an atomic force microscope for force spectroscopy and imaging to analyze the formation of networks upon mixing various protein constructs. We find that small scale networks form using both Streptavidin and SpyTag-SpyCatcher as crosslinkers and that SpyCatcher can be used to detect molecular crosslinking and polyprotein polarization through SMFS.

Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.

In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.

The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.