Projects
More projects will soon be available
Imaging Single Serotonin Receptors: Advisor Prof. J. A. Brozik
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Fluorescence image from a single pentameric 5HT3R receptor labeled at the C-terminus of each subunit in DMPC at 0o C.
Fit of top image to a point spread function (PFS) locating the peak to 1.5nm accuracy. As each label bleaches or blinks the PSF will wobble. This ‘wobble’ reflects the topography of the receptor. In this way we can begin to map out the shape of the receptor (currently we have determined the receptor to have a diameter of ~10.8nm.) |
About 20% of the proteins in any given organism are classified as integral membrane proteins. For example, of the 4280 proteins that make up E. coli, 849 are membrane proteins5. Given these large numbers, it is remarkable to learn that, to date, there are only about 150 unique membrane protein structures (including proteins of the same type but from different species) that have been determined through X-ray crystallography, electron diffraction, or NMR spectroscopy. Moreover this class of proteins account for some incredibly important and diverse cellular function; including ion regulation and transport, molecular recognition and response, and energy transduction. In many ways the lack of structural information has been the major obstacle in developing fundamental models detailing how this important class of proteins function. The reasons for the lack of structural information are many and varied, but the main contributors are (1) the difficulty of producing membrane proteins in high concentrations (they only exist in the very thin membranes surrounding the cell and the organelles of the cell) and (2) the fact that cell membranes are integral parts of their structure and are often needed for correct folding and assembly.
By their very nature, membrane proteins are stochastic and can diffuse around in supported lipid membranes. Such dynamic behavior can be very informative and is fundamental in its own right, but it presents a major technical hurdle when trying to determine information about protein structure. To overcome this problem we have recently developed a cryogenic single molecule experiment and have begun to map out the topography of the serotonin type-three receptor (5HT3A); see figure 1. While this series of experiments are quite important in their own right; the dynamic (and therefore thermodynamic) information associated with intra-protein motion is completely frozen out. In collaboration with Dr. George Bachand and Dr. Gabriel Montano at the Center for Integrative Nanotechnology (CINT), we have (and still are) bioengineering a series of 5HT3A mutants that have anchors in their intercellular loops in order to orient and immobilize these receptors to suitable optical substrates. What I would like an REU student to do is to use one of these new bioengineered 5HT3A receptors and build a biomimetic assembly embedded into a lipid bilayer membrane attach to an optical substrate in such a way that it remains functional. Once that has been achieved, I would like that student to make the initial physical measurements probing intra-protein fluctuations at ambient temperatures and in a fluid membrane (a more natural environment for probing the machine-like properties of membrane proteins) using single molecule microscopy / spectroscopy.
Biochemical and Biophysical Studies of Calsequestrin: Advisor Prof. C. H. Kang
The sarcoplasmic reticulum (SR) plays an essential role in excitation-contraction coupling by regulating the cytosolic Ca2+ concentration. One of the main SR proteins responsible for this regulation is the Ca2+ storage protein, calsequestrin (CSQ). A CSQ molecule binds 40-80 Ca2+ ions, thus acting as a Ca2+ buffer and regulating the amount of Ca2+ released from both cardiac and skeletal SR. Over the last several years, an increasing number of sudden arrhythmia, catecholamine-induced polymorphic ventricular tachycardia (CPVT), has been attributed to mutated CSQ, which currently has a very high mortality rate (35%). In our previous research, we discovered a unique coupling mechanism between ion-dependent polymerization of CSQ and its high capacity Ca2+ binding. In mutated CSQs of CPVT patients, altered behavior in both polymerization and Ca2+ binding capacity were detected. We also found that CSQ has putative ligand-binding sites, and small molecules appear to interfere with the Ca2+ binding capacity of CSQ by occupying these sites. These interfering small molecules include anthracycline anticancer drugs, phenothiazine antidepressants and several other compounds, all of which have muscle-related side effects including tachycardia. The physiological and biomedical relevance of this discovery is high and provides a compelling case for the continuation of our studies. The focus of our investigation is now to carry out biophysical and biochemical studies to understand the molecular details of the unique Ca2+ regulation mechanism by CSQ and other components of the Ca2+ channel complex, as well as, study CSQ‟s altered Ca2+ regulation in CPVT. At the same time, we will investigate the molecular mechanism by which pharmaceutical drugs interfere the Ca2+ regulation of CSQ and SR. Therefore, a perspective REU student will work with the PI and the rest of the research team to innovatively establish the potential linkage between cardiac complications of CSQ affinity drugs and the CPVT caused by mutated CSQs. These studies will improve our understanding of the regulatory mechanisms involved in Ca2+ cycling by both cardiac and skeletal muscles and of the adverse effects of some pharmaceutical drugs on the muscle. It will also provide an essential step in the development of various therapeutic agents with minimal side effects and effective treatments for some hereditary heart diseases such as CPVT.
Ion Mobility Mass Spectrometry (IMS and IMMS) of Metabolites: Advisor Prof. H. Hill
We are involved in the fundamental development of an analytical method known as ion mobility spectrometry (IMS) and ion mobility spectrometry coupled with mass spectrometry (IMMS). Our research investigates ion chemistries, ion transport processes in buffer gases and liquids, and ion detection processes. Of particular interest are the ion chemistries and transport properties at high pressures and ion chemistries and transport properties in non-electrolyte containing liquids. Various novel ionization processes are currently under investigations including, 1) secondary electrospray ionization, 2) dispersive corona discharge ionization and 3) electro-dispersive ionization. Current application goals are for the identification of oligosaccharides structures using ion mobility mass spectrometry, the identification and quantification of metabolites in blood samples, the separation of isomers, conformers and chiral compounds, the identification of interferences for explosive and chemical warfare agent detectors.
Such a wide range of projects lead to a number of specific experiments suitable for undergraduate research. Calibration, development of databases, comparison of theory and experiment, kinetics, etc. Of particulate interest at the moment is the careful calibration of mobility values with respect to temperature and impurities in the buffer gas. Using an ion mobility time-of-flight mass spectrometer which has been developed in our laboratory, we propose that an undergraduate would evaluate the effects of temperature and humidity on the mobility of ions. Currently ion mobility can be measured to three significant figures, the goal of this project is to extend the calibration of IMS to four significant figures and thus be able to measure the size of ions to a higher accuracy. These studies will provide an undergraduate with instrumental experience in ion mobility and mass spectrometry and will acquaint him or her with gas phase theory of ion transport processes.
Molecular Design of Probes for Prostate Cancer: Prof. P. Benny
A student project would involve a fundamental study of cell interactions with non-steroidal anti-androgens compounds. The project goal would be to design compounds that incorporate an imaging moiety (i.e., photoactive, radiolabel, near IR) to gain quantitative information about the uptake, localization, and metabolism of the prepared compounds in prostate cancer cells. The individual student training would involve learning and utilizing a number of cross-disciplinary techniques from chemistry and biology. The project would involve the synthesis and characterization of model compounds, which would involve a combination of organic, inorganic and radiochemistry experiments. The compounds would be further examined by in vitro testing focusing on determining the location and transportation mechanism across the cellular membrane and nuclear envelop. A student would utilize several prostate cancer cells lines (PC3, LnCap, DU-145) to compare the imaging uptake and localization in the cells. Each of these cell lines represents broad classes of cancer types with specific exocellular markers for targeting. Overall, the student project would involve a multidisciplinary experience integrated to specific understand and measure compound in and across cell membranes.
Model Protein Systems in Solution and on Surfaces: Advisor Prof. U. Mazur
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Protein aggregates and the process of protein aggregation itself has attracted considerable interest recently, because they are associated with a large number of neurodegenerative diseases including Alzheimer‟s, Parkinson‟s, and prion diseases6-9. The aggregates form after an onset of destabilization of the native protein. The source of the destabilization can be mutation or an environmentally induced alteration resulting in an intermediate structure with marginal stability (a misfolded protein)8, 9. The past decade has seen numerous theoretical and experimental advances in our understanding of protein folding10-12. However, from the perspective of aggregation, it is crucial to know how the proteins actually aggregate and disaggregate in order to understand the mechanism of this process and thus more finely tune the design of antiaggregating agents. Understanding the mechanism of protein aggregation consists of knowing the structures, energetics, and dynamics of these organized assemblies.
The REU students in this research group will study the aggregation of proteins in solution and adsorbed on surfaces by utilizing Scanning Probe Microscopy and optical and electronic spectroscopy. To this end we plan to employ heme peptide model systems. Our approach to investigating aggregation is unique in that we have discovered that it is possible to do sub-molecular imaging of these aggregates when adsorbed on a metal substrate. Furthermore, different ligands produce different aggregate structures that are easily differentiated by STM. Previous STM studies did not produce sub-molecular images nor have they demonstrated the ability to differentiate aggregation state12-16.
The adjacent figure shows our recent very dramatic STM images obtained from microperoxidase-11, MP-11, adsorbed from different fluid solution environments on Au(111)17. (To our knowledge this quality of resolution has not been previously observed for any complex biological system). When the Au substrate is exposed to a solution containing MP-11 and imidazole we observe regular elongated molecular or chains (a). These molecular stacks are most likely the result of electrostatic interactions between one or more MP-11 molecules assisted by ferric heme-imidazole interactions. In figure (c) we observe single isolated molecules of MP-11. Here, it‟s most likely that the added sulfide dianion bonds directly to the iron center of the heme and subsequently to the gold surface upon adsorption from solution. We have demonstrated that we can, in fact, resolve submolecular features of the protein chain laying over the ferric porphyrin moiety, as seen in the inset of figure (c). The „high spot‟ in that molecular image coincides well with the location of increased electron density. Microperoxidases (MP) are good peptide model systems for us to exploit for this project because they are such a well-studied class of metalloporphyrin-peptides derived form natural heme proteins. MP-8, which we also will utilize in our studies, contains only 8 amino acid residues with an overall more negatively charged surface than MP-11. With the MP-8 system we expect to observe a different aggregation behavior than with MP-11. The small size of the peptide chains in MP-11 and MP-8, and the availability of the porphyrin ring which can be easily recognized in STM images,18, 19 offers us the best chance to examine aggregation (and potentially even peptide structure) on a molecular level (under ambient conditions or in solution under potential control). Obtain high resolution images of MP-11 and MP-8 complexed with imidazole, small amino acids, and ions (S2, N3, SCN), by STM both in ambient environment and in solution to better resolve the structural details of the aggregates and understand the mechanism of formation. We will also study the effects of MP-11 (and MP-8) to ligand ratio, solution concentration, solvent, pH, and temperature on the aggregation process and aggregate structures. Map out conductance pathways in the different MP-11 and MP-8 aggregates in order to better understand electron transport in these systems by utilizing scanning tunneling spectroscopy. These studies will also assist us in identifying binding sites in aggregates. Conduct simultaneous optical measurements (UV-vis) on the MP-11 (and MP-8)/ligand systems to ascertain the degree of association in solution and to determine oxidation and ligation states of the iron porphyrin.
Preparation of the biotin probe for S-nitrosylated proteins: Advisor Prof. M. Xian
| Protein S-nitrosylation, i.e. converting cysteine residues (-SH) to S-nitrosothiol adducts (-SNO), is a post-translational modification elicited by nitric oxide (NO). This biological reaction has a broad spectrum of effects in physiology. Monitoring the formation of S-nitrosothiols in living systems is vital to understand NO signal transduction. However, the detection and quantification of protein SNO modification still remains challenging because of the lability of the SNO group. Recently, our group developed a new bioorthogonal reaction which specifically targets S-NO groups. This reaction can convert unstable S-nitrosothiols (A, Scheme 1) to stable sulfenamide products B in one step. Based on this strategy, we can design and prepare biomarkers for labeling SNO proteins. |  |
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A summer student involved in this project is expected to carry out the synthesis of a biotin probe for SNO proteins based on the new reaction. The target, i.e. compound 1 (Scheme 2), will be prepared as the following: Pd-catalyzed cross coupling between commercially available starting materials 2 and Ph2PH will provide compound 3. This compound will then be subjected to standard amide formation with compound 4 to furnish compound 7. Compound 4 is expected to be prepared from biotin-acid 5 and diamine 6, again under standard amide formation conditions. Finally, compound 7 will be converted to the target molecule 1 in two steps: LiOH mediated hydrolysis and phenyl ester formation. With compound 1 in hand, the student will collaborate with other group members to evaluate its efficiency as SNO protein marker (Scheme 2). |
Photoswitchable Nanoparticles for Live Cell Imaging: Advisor Prof. Alex Li
In professor Li‟s labs, research will focus on using new nanoparticles containing a single dye but emits two fluorescence colors. The major efforts will be synthesizing and characterizing optically switchable nanoparticles, which were constructed following our previously reported procedure to incorporate various photochromic spiropyran derivatives into the hydrophobic cores of the nanoparticles20, 21. Typically, major monomers acrylamide (A), styrene (ST), and butyl acrylate (BA) are polymerized with minor functional monomers, including optically switchable spiropyran derivatives, the cross-linker divinyl benzene (DVB), and water-soluble acrylic acid (AA) decorating the nanoparticle surfaces. The nanoparticles produced range from 60 to 80 nm as characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The resulting nanoparticles will be decorated with high mobility group A proteins (HMGA) and we will monitor the binding and drifting and finally endocytosing such dual color nanoparticles into live cells. Previously dual color photoswitching was achieved using FRET between two dyes: one photochromic spiropyran and another fluorescence donor chromophore. Now, the REU students will be experimenting a single spiropyran derivative that exhibits dual-color photoswitching in nanoparticles and one possible module will be synthesizing such spiropyran dyes. After synthesizing nanoparticles containing the desired photoswitchable dye, the REU student will be trained to characterize their sizes using TEM and DLS. Using the TEM and DLS as calibration tools, the student will learn to prepare nanoparticles with narrow and uniform distribution. Once mastering the nanoparticle synthetic techniques, another REU student will proceed to photo- chemical and physical studies of the photoswitchable-dye containing nanoparticles. In these processes, students will learn not only synthetic skills, but also fundamental aspects of molecular structures, electronic structures, molecular spectroscopy, reaction kinetics, chemical equilibrium, quantum chemistry, and light-matter interactions. Thus, the lucky student will benefit tremendously from this NSF-funded REU program.