Browsing by Subject "protein folding"

As of late 2009, the Protein Data Bank (PDB) has grown to contain over 70,000 models. This recent increase in the amount of structural data allows for more extensive explication of the governing principles of macromolecular folding and association to complement traditional studies focused on a single molecule or complex. PDB-wide characterization of structural features yields insights that are useful in prediction and validation of the 3D structure of macromolecules and their complexes. Here, these insights lead to a deeper understanding of protein--protein interfaces, full-atom critical assessment of increasingly more accurate structure predictions, a better defined library of RNA backbone conformers for validation and building 3D models, and knowledge-based automatic correction of errors in protein sidechain rotamers.

My study of protein--protein interfaces identifies amino acid pairing preferences in a set of 146 transient interfaces. Using a geometric interface surface definition devoid of arbitrary cutoffs common to previous studies of interface composition, I calculate inter- and intrachain amino acid pairing preferences. As expected, salt-bridges and hydrophobic patches are prevalent, but likelihood correction of observed pairing frequencies reveals some surprising pairing preferences, such as Cys-His interchain pairs and Met-Met intrachain pairs. To complement my statistical observations, I introduce a 2D visualization of the 3D interface surface that can display a variety of interface characteristics, including residue type, atomic distance and backbone/sidechain composition.

My study of protein interiors finds that 3D structure prediction from sequence (as part of the CASP experiment) is very close to full-atom accuracy. Validation of structure prediction should therefore consider all atom positions instead of the traditional Calpha-only evaluation. I introduce six new full-model quality criteria to assess the accuracy of CASP predictions, which demonstrate that groups who use structural knowledge culled from the PDB to inform their prediction protocols produce the most accurate results.

My study of RNA backbone introduces a set of rotamer-like "suite" conformers. Initially hand-identified by the Richardson laboratory, these 7D conformers represent backbone segments that are found to be genuine and favorable. X-ray crystallographers can use backbone conformers for model building in often poor backbone density and in validation after refinement. Increasing amounts of high quality RNA data allow for improved conformer identification, but also complicate hand-curation. I demonstrate that affinity propagation successfully differentiates between two related but distinct suite conformers, and is a useful tool for automated conformer clustering.

My study of protein sidechain rotamers in X-ray structures identifies a class of systematic errors that results in sidechains misfit by approximately 180 degrees. I introduce Autofix, a method for automated detection and correction of such errors. Autofix corrects over 40% of errors for Leu, Thr, and Val residues, and a significant number of Arg residues. On average, Autofix made four corrections per PDB file in 945 X-ray structures. Autofix will be implemented into MolProbity and PHENIX for easy integration into X-ray crystallography workflows.

Understanding the interconversion between the thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was used as a model system to elucidate the general folding/unfolding of an intrinsically unstructured protein (IUP) both in the absence and presence of ligands.

P protein was previously characterized as an intrinsically unstructured protein, and it is predominantly unfolded in the absence of ligands. Addition of small anions can induce the protein to fold. Therefore, the folding and binding are tightly coupled. Trimethylamine-N oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic time courses at various final TMAO concentrations showed multiphase kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea to obtain a TMAO-urea titration surface of P protein. Both kinetic and equilibrium studies show evidence of an intermediate state in the P protein folding process. The intermediate state is significantly populated and the folding rate constants involved in the reaction are slow relative to similar size proteins.

NMR spectroscopy was used to characterize the structural properties of the folding intermediate of P protein. The results indicate that the N-terminal (residues 2-19) and C-terminal regions (residues 91-116, 118 is the last residue) are mostly unfolded. 1H-15N HSQC NMR spectra were collected at various pH values. The results suggest that His 22 may play a major role in the energetics of the equilibria between the unfolded, intermediate, and native states of P protein.

Ligand-induced folding kinetics were also investigated to elucidate the overall coupled folding and binding mechanism of P protein and the holoenzyme assembly process. Stopped flow fluorescence experiments were performed at various final ligand concentrations and the data were analyzed using a minimal complexity model that included three conformational states (unfolded, intermediate and folded) in each of three possible liganding states (0, 1 and 2 ligands). The kinetic and equilibrium model parameters that best fit the data were used to calculate the flux through each of the six possible folding/binding pathways. This novel flux-based analysis allows evaluation of the relative importance of pathways in which folding precedes binding or vice versa. The results indicate that the coupled folding and binding mechanism of P protein is strongly dependent on ligand concentration. This conclusion can be generalized to other protein systems for which ligand binding is coupled to conformational changes.

Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Unfortunately, these studies have not been as useful as expected at identifying disease-related proteins that can be exploited for diagnostic and therapeutic purposes, presumably due to the indirect link between a protein’s expression level and its function. Investigated here is the use of thermodynamic stability measurements to probe a more biologically relevant dimension of the proteome. It has the potential to become a new strategy for disease state characterization and to help elucidate the molecular basis of the disease. This thesis outlines the use of two discovery based techniques and one validation based technique to study protein folding and stability changes associated with breast cancer.

The first part of this dissertation describes the application of a mass spectrometry-based technique, stable isotope labeling with amino acids in cell culture and stability of proteins from rates of oxidation (SILAC-SPROX), in a comparison of the MCF-7 versus BT-474 breast cancer cell lines and a comparison of the MCF-7 versus MDA-MB-468 breast cancer cell lines. This work enabled ~1000 proteins to be assayed for breast cancer-related thermodynamic stability differences. The 242 and 445 protein hits identified with altered stabilities in these comparative analyses created distinct molecular markers to differentiate the three cell lines.

The second part of this dissertation describes the development of a SILAC-based limited proteolysis (SILAC-LiP) strategy. The applicability of the protocol was demonstrated in a proof-of-principle study using proteins from a yeast cell lysate and a ubiquitous ligand. The SILAC-LiP protocol was further applied in a comparison of the MCF-7 versus MCF-10A cell lines. This work identified ∼200 proteins with cell line dependent conformational changes, as determined by their differential susceptibility to proteolytic digestion using the nonspecific protease, proteinase K. The overlap between the SILAC-LiP hits reported here and the SILAC-SPROX hits previously identified in these same cell lines was relatively small (~20%). Thus, this work indicates that the SILAC-SPROX and SILAC-LiP techniques can be used together to provide complementary information on the disease states.

Furthermore, the protein hits identified in both the SILAC-SPROX and SILAC- LiP experiments included a large fraction (∼70%) with no significant expression level changes. This suggests protein folding and stability measurements can provide information about disease states that is orthogonal to that obtained in protein expression level analyses.

The last part of this dissertation focuses on the establishment of targeted mass spectrometry-based validation assays for the protein biomarker candidates with altered thermodynamic stabilities identified in the SILAC-SPROX experiments. Application of the PAB-SPROX protocol on the MCF-7 cell lysate enabled reproducible identification and quantitation of a subset of prioritized target peptides.

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.

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.