CHEM 645 Protein Structure and Function Schedule


Class Schedule Target to Current Class - last updated 12/7/2011 Wed 8/31/11 - Our first class will be next Wednesday, due to the delayed start of UD classes. But please read below and come to class prepared to review protein structure. assignment for next class: Review any biochemistry textbook description of primary, secondary and tertiary structure of proteins. Also read Chapter 1 of Crystallography Made Crystal Clear (CMCC). In case you haven't purchased the text yet, you can get a pdf of this chapter by clicking here. Be prepared to come to the board to participate in the review. Wed 9/7/11 - Class introduction and protein structure review Class introduction In class, we will go over the syllabus from the course web page, including tentative schedule, homology modeling problem set description and your class presentations. Then we will go around the room to introduce ourselves to each other. Then we will watch a documentary that aired on PBS that presents an interesting view of graduate school in the Shapiro lab at Columbia, which uses structural biology. It's titled "Naturally Obsessed" http://naturallyobsessed.com/blog/see-the-film/ Protein structure review We reviewed a protein's primary structure, peptide planes, bond angles, bond lengths, torsion angles, phi-psi plots, RMSD as a criteria of protein geometry and knowledge of how basic protein structure decreases the number of parameters varied in a refinement. Then I introduced some ideas needed to understand protein refinement. web sites visited in class: Chem 645 class web page Download the a pdf file protein-review.pdf of the class notes. MPG movie shown in class assignment for next class: Chapter 3 from Crystallography Made Crystal Clear (CMCC). The next class will focus on sample preparation and the need for homogeneity for protein crystal growth, which is covered in chapter 3 of CMCC. Come to class prepared to share in the discussion (see below) using either your personal research experiences in producing homogeneous protein samples (and possibly crystal growth attempts) or from a recent paper or web site that addresses some of these issues. For example, you could focus here on techniques used to purify or techniques used to analyze homogeneity, such as isoelectric focussing gel electrophoresis (IEF), dynamic light scattering (DLS), etc... Wed 9/14/11 - Protein production - homogeneity/heterogeneity In part one of the class, meet in 221 BRL We introduced how proteins pack into a crystal through "crystal contacts" to form a lattice in a protein crystal. Then we talked about homogeneous protein samples: how to assess, how to improve, and related this to the ability to grow protein crystals. Everybody in class will contribute to this discussion from personal research experience or from a recent paper or web site that addresses these issues (protein purity, homogeneity/heterogeneity, protein crystallization). PDF of Powerpoint in class homogeneity-xtal-growth.pdf web sites related to class: A site describing crystal contacts - http://molvis.sdsc.edu/protexpl/xtlcon.htm Hampton Research - the company that sells protein crystallization supplies In part two of the evening, we will then go on a "field trip to the Bahnson Lab" (312-314 Drake Hall) to setup lysozyme crystals. assignment for next class: Chapter 2 from CMCC, which is an intro to crystallography, then read Chapter 4 from CMCC, which covers "geometric principles and diffraction". Wed 9/21/11 - X-ray equipment, diffraction and reciprocal space In part one of the class, meet in 221 BRL We briefly saw how x-rays are generated, either on an in-house rotating anode generator or at a synchrotron. The data collection process was described. Because X-rays cannot be focussed like visible light we need to know the wavelength, amplitude and relative phase of each reflection. The wavelength is controlled by the source of x-rays. The relative amplitudes can be measured in a straightforward manner and the phase is the tricky part (see next class). Reflections are the result of constructive interference of diffraction off of parallel planes running through the crystal. Important concepts related to this are: Miller indices - hkl, reciprocal vs. real space, Bragg's Law. In part two of the evening, we will once again reconvene in 312-314 Drake Hall and collect some data on the crystals we grew last week and also look at some molecular models with electron density maps on a graphics terminal. web sites related to class Download the PDF of the POWERPOINT diffraction.pdf used in class. assignment for next class: Chapters 5-7 from CMCC Wed 9/28/11 - Phasing and model building Download the pdf phases-model-building.pdf In part I we went over the relationships that describe real space and reciprocal space in terms of electron density (real space) and structure factors (amplitude, frequency and phase of reciprocal space). Methods used to obtain phases were described. Multiple isomorphous replacement (MIR phasing) involve introducing heavy atoms (Hg, Pt, Ur, etc.) into a protein crystal and accurately measuring changes. In the 2nd half we discussed Molecular Replacement (MR) and MAD phasing (and SAD phasing). We then went through steps involved in building a structure, such as solvent flattening, defining molecular boundaries, defining non-crystallographic symmetry axis, building the main chain trace, alpha-helices, beta-turns, adding side-chains. Next, this was all tied into refinement, adjusting the model and finishing up a structure. assignment for next class: CMCC, pgs 259-268 of Chapter 10.3. Homology Models + Other Theoretical Models, also look at some of these links: Swiss Model - http://swissmodel.expasy.org//SWISS-MODEL.html NCBI PubMed - http://www.ncbi.nlm.nih.gov/sites/entrez/ Homology modeling tutorial - http://molvis.sdsc.edu/protexpl/homolmod.htm Principles of Protein Structure, Comparative Protein Modeling and Visualization (http://swissmodel.expasy.org//course/course-index.htm) Also, as mentioned in class, bring laptops to the next class and preload pymol, deepview and a pdb file with more than one chain. Also edit the pdb file to include only chain A. Wed 10/5/11 - Homology modeling introduction Tonight, we introduced homology modeling in general, and then used the example of a previous year's modeling assignment (human ALR) to show how its done. This is the first introduction to your modeling project. Click Here for part-I of your modeling assignment of the protein NCEH1. websites visited in class: Swiss Model - http://swissmodel.expasy.org//SWISS-MODEL.html NCBI PubMed - http://www.ncbi.nlm.nih.gov/sites/entrez/ Deepview - http://spdbv.vital-it.ch/ PyMol - http://www.pymol.org Protein Data Bank - www.pdb.org ExPASy (http://us.expasy.org/) - many usefull programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) -generates multi-sequence alignments BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html) - takes output from Clustalw and makes nice figure. assignment for next class: Chapter 10 from CMCC, pages 237-259, Macromolecular NMR. Also download class notes (below) and visit the website: - The Basics of NMR http://www.cis.rit.edu/htbooks/nmr/inside.htm Wed 10/12/11 - Introduction to NMR Download the pdf intro-NMR.pdf In class we went over the basics of NMR, focusing on 1-dimensional proton NMR. The layout of an NMR spectrometer was described along with what happens to the nuclei during a 90 deg. pulse and data acquisition (FID). Through bond (aka scalar and J-coupling) was introduced, which is the basis of a COSY 2-D experiment. Through space coupling (dipolar) was described as the basis of the Nuclear Overhauser Effect (NOE), which we'll see in the NOESY 2-D experiment. assignment for next class: Wuthrich, (1989) Science 243, 45-50. Ikura et al., (1989) Biochemistry 29, 4659-4667. A 2nd review: Wuthrich, (1990) J. Biol. Chem. 265, 22059-22062. Also, you should turn in part-I of modeling assignment during next class. Wed 10/19/11 - 2-D and 3-D NMR methods and macromolecular NMR Download the pdf part-II-NMR.pdf We reviewed 1-D and 2-D briefly and discussed how proteins less than 10 kDa can be solved using these methods. Then I discussed heteronuclear 3-D and 4-D techniques, drawing from the paper by Ikura et al., 1989. Finally we looked at some of the significant differences between NMR and Crystal structures. You should turn in part-I of modeling assignment tonight. here is a pdf file of a previous year's midterm Wed 10/26/11 - In class midterm, open notes and open book Wed 11/2/11 - Presentations discussion and NCEH1 part II The second part of our NCEH1 project was assigned tonight, which includes a little bit of protein design, molecular biology and functional questions. Then we discussed presentation topics and hopefully picked dates for everyone. click here for the NCEH1-part II assignment Wed 11/9/11 - Protein Aggregation and Misfolding Disorders Presenting tonight: Na Young, Ben and Chris 1) Na Young will present topic related to "Perturbation of the Stability of Amyloid Fibrils through Alteration of Electrostatic Interactions" Shammas et al. (2011) Biophysical Journal 100, 2783–2791. click here 2) then Chris will present topic related to "a-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation" Bartels et al. (2011) Nature, 447, 107-111. click here 3) and last, but not least, Ben will present topic related to "Sequence-independent Control of Peptide Conformation in Liposomal Vaccines for Targeting Protein Misfolding Diseases", Hickman et al, (2011) J. Biol. Chem., 286, 13966-76. click here Wed 11/16/11 - Therapeutics: proteins, inhibitors, etc... Presenting tonight: Yiben, Steve, Abhijit and Devin 1) Yiben will start with a brief background on protein therapeutics 2) Steve will present a paper on immunogenicity "Reducing risk, improving outcomes: Bioengineering less immunogenic protein therapeutics" Groot and Martin (2009) Clinical Immunology, 131, 189-201 click here 3) Abhijit will present a paper on drug design "Rational Drug Design", Mandal et al (2009) Eur. Journal of Pharm. 625, 90-100. click here 4) Devin will present compare and contrast two papers on drug delivery "Liquid filled nanoparticles as a drug deliverytool for protein therapeutics" Venkatesan et al (2005) Biomaterials 26, 7154-7163. click here "Systemic Delivery of Secreted Protein b y Grafts of Epidermal Keratinocytes: Prospects for Keratinocyte Gene Therapy" Fenjves et al (1994) Human Gene Therapy 5, 1241-48. click here 5) Yiben will wrap it up with a presentation of emerging trends "Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells" Grillberget et al (2009) Biotechnology Journal 4, 186-201. click here Wed 11/23/11 - No class, night before Thanksgiving. Wed 11/30/11 - Protein Folding Presenting tonight: Lingxi, Tingwei, Ping and Wei 1) Lingxi will present on oxidative protein folding "Oxidative Protein Folding in Vitro: A Study of the Cooperation between Quiescin-Sulfhydryl Oxidase and Protein Disulfide Isomerase" Rancy and Thorpe (2008) Biochemistry, 47, 12047-56 click here 2) Ping will present a paper on single molecule work "Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein" Pirchi et al. (2011) Nature Communications, 2:43, 1-7 click here 3) Tingwei will present a paper on multiphoton ANS Fluorescence work "Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress", Hadley et al (2011) Cell Stress and Chaperones, 16, 549-561. click here 4) Wei will present a paper on the latest approach at predicting protein folding "Predicting protein structures with a multiplayer online game", Cooper et al. (2010) Nature 466, 756-760. click here Also NCEH1-part-II is due tonight Wed 12/7/11 - Protein Dynamics Presenting tonight: Tim, Chris and Tiffany 1)Tim will present on a 2-photon approach to follow protein dynamics in cells "Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time" Martini et al. (2007) Journal of Structural Biology 158, 401–409 click here 2) Chris will present an overview of how SAXS helps sort out dynamics in protein assemblies "Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering" Rambo and Tainer (2010) Current Opinions in Structural Biology, 20, 128-137 click here 3) And in our last presentation of the semester, Tiffany will present a paper titled: "Protein Dynamics in Drug Combinations: a Linear Superposition of Individual-Drug Responses", Geva-Zatorsky et al. (2010) Cell, 140, 643-651 click here Thur 12/8/11 - Take home final assigned On this day, you can click here to get a WORD version the exam and click here for a pdf version of the exam. Your finished exam must be e-mailed to me by 11:59 pm, Wednesday 12/14/2011. click here for interesting class photo Course Evaluations Between Friday, December 2nd through midnight of Thursday, December 8th, please fill out course evaluations (http://www.udel.edu/course-evals). Back to the Chem 645 home page Chemistry & Biochemistry home page