Browsing by Subject "ligand binding"
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Item Open Access Aqueous Desolvation and Molecular Recognition: Experimental and Computational Studies of a Novel Host-Guest System Based on Cucurbit[7]uril(2012) Wang, YiMolecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.
In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.
We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.
To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.
The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.
Item Open Access Development and Application of a Mass Spectrometry-Based Assay for the High Throughput Analysis of Protein-Ligand Binding(2009) Hopper, Erin D.Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.
This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.
The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.
To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.
Item Open Access Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding(2016) Xu, YingrongThermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.
Item Open Access Novel Algorithms for Computational Protein Design, with Applications to Enzyme Redesign and Small-Molecule Inhibitor Design(2009) Georgiev, Ivelin StefanovComputational protein design aims at identifying protein mutations and conformations with desired target properties (such as increased protein stability, switch of substrate specificity, or novel function) from a vast combinatorial space of candidate solutions. The development of algorithms to efficiently and accurately solve problems in protein design has thus posed significant computational and modeling challenges. Despite the inherent hardness of protein design, a number of computational techniques have been previously developed and applied to a wide range of protein design problems. In many cases, however, the available computational protein design techniques are deficient both in computational power and modeling accuracy. Typical simplifying modeling assumptions for computational protein design are the rigidity of the protein backbone and the discretization of the protein side-chain conformations. Here, we present the derivation, proofs of correctness and complexity, implementation, and application of novel algorithms for computational protein design that, unlike previous approaches, have provably-accurate guarantees even when backbone or continuous side-chain flexibility are incorporated into the model. We also describe novel divide-and-conquer and dynamic programming algorithms for improved computational efficiency that are shown to result in speed-ups of up to several orders of magnitude as compared to previously-available techniques. Our novel algorithms are further incorporated as part of K*, a provably-accurate ensemble-based algorithm for protein-ligand binding prediction and protein design. The application of our suite of protein design algorithms to a variety of problems, including enzyme redesign and small-molecule inhibitor design, is described. Experimental validation, performed by our collaborators, of a set of our computational predictions confirms the feasibility and usefulness of our novel algorithms for computational protein design.
Item Open Access Proteomic Methods and Applications for Protein Folding Stability Measurements(2019) Meng, HeIn the last 10 years, several mass spectrometry-based proteomic techniques have been developed for the large-scale characterization of protein conformations, thermodynamic stabilities, and protein−ligand interactions. The main focus of this dissertation involves the development and application of several mass spectrometry-based-methods in this current suite of proteomics techniques for the large-scale analysis of protein folding and stability measurement. One goal of this work is to investigate the use of protein folding and stability measurements to better detect and understand the biophysical properties of post-translational modifications. Another goal of this work is to develop a novel protein stability measurement technique for making thermodynamic measurements of protein folding and ligand binding interactions. This technique, which involves a combination chemical denaturant and protein precipitation yields significantly better proteomic coverage and a largely reduced false discovery rate compared to its sister technique, Stability of Proteins from Rates of Oxidation (SPROX).
The first part of this dissertation describes the application of the stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) on comparing the conformational properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. A total of 168 and 251 protein hits were identified with dephosphorylation-induced stability changes using the SPROX and LiP techniques, respectively. The SPROX results revealed that the magnitudes of the destabilizing effects of dephosphorylation on the different aaRSs were directly correlated with their previously reported aminoacylation activity change upon dephosphorylation. The example of these aaRSs substantiates the close link between protein folding thermodynamic and function and helps establish the utility of thermodynamic stability measurements for understanding protein function.
The second part of this dissertation describes the development of a new protein-stability based proteomic method for identification and quantification of protein-drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔGf) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein−drug binding affinities. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast lysate. The technique was also used to identify protein targets of sinefungin in a human MCF-7 cell lysate. The CPP technique yielded dissociation constant (Kd) measurements for these well-studied drugs that were in general agreement with previously reported Kd or IC50 values.
The third part of this dissertation describes two protein target discovery applications of the CPP approach including one involving subglutinol A (a natural product with immunosuppressive activity) and one involving clemastine fumarate (an existing anti-histamine drug with recently discovered anti-malarial activity). As part of this work, about 800 proteins in a mouse 2B4 T cell lysate were assayed for subglutinol A-induced stability changes, and deoxycytidine kinase was identified as the protein hit. The magnitude of the ligand induced stability change was used to calculate a Kd value of 250 M, which is close to the reported cell based IC50. In the protein target discovery study on clemastine fumarate, a total of 800 yeast proteins were assayed for drug-induced stability changes and 8 protein with clemastine-induced stability changes were identified, including SEC14 cytosolic factor, glycerol kinase, asparagine--tRNA ligase, and inosine triphosphate pyrophosphatase. The latter two applications demonstrate that CPP can reliably identify and quantify protein-drug interactions in a complex biological mixture, making it a valuable addition to the current suite of proteomic methods for the large-scale detection and quantitation of protein-ligand binding interactions.