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Previous Classes Guidelines Previous Classes PowerPoint Presentations
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Special Research Project for Fall 2011 A Historical Perspective: Simply put, our investigations have attempted to look at the effects of temparture and three reducing agents (BME, DTT, and TCEP) on enzymatic activity and structural integrity of AP. At the most fundamental level, the question we are asking is how much importance do the two disulfide bonds with the native enzyme have on these properties. Before considering AP further, a brief look at the legendary experiments performed by Christian Anfinsen on the denaturation and refolding of ribonuclease A in the 1950’s (which led to a Nobel Prize) might provide us with some insights. Anfinasen’s research is summarized very well in the Journal of Biological Chemistry Classics article RNase1.pdf and the related Haber and Anfinsen paper RNase2.pdf, which you are strongly advised to read. Another explanation of Anfinsen’s experiments is outlined in Lehninger Principles of Biochemistry (pps. 140 – 142). These landmark experiments provided strong evidence for the notion that the folded, or tertiary structure, of a protein is primarily determined by the amino acid sequence and the proclivity those amino acids have to form secondary structures, which are believed to act as a nucleation sites along the folding pathway (see pps. 142 – 143 in Lehninger) for collapse of the protein to its native tertiary structure. We must be careful, however, to realize that in Ainfensen’s experiments an important procedure they carried out involved the inclusion of urea as a general protein denaturant, which in our experiments has not used. A more thorough explanation of the relationship between Ainfensen's studies and our work with AP can be read in an article we published in the online journal Enzymatic in May, 2010: JBC Classics: Standing on the Shoulders of Giants! In a manner similar to ribonuclease, E. coli alkaline phosphatase contains two disulfide bonds per monomer which are undoubtedly related to its structural stability and its activity (see below Sone et. al, 1997). Structural stability is undoubtedly very important for this protein because it is located within the periplasmic space in which the chemical environment can be highly variable. This project was originally based upon two important experiments or observations. First, earlier in the semester enzyme assays were performed on native PAGE gels, attempting to see enzyme activity for β−galactosidase and alkaline phosphatase (AP) using two different colorimetric reagents. The results from these experiments allowed for the development of a hypothesis which provided us with a convenient platform from which to ask important questions about AP as well as the way in which the native gel assay was performed. Second, an important feature about AP that needs to be considered is that during its purification, you heat the periplasmic lysate to 80 C for 15 minutes, resulting in the denaturation of many proteins that are not as thermally stable as AP. The logical question from this fact is what makes AP so thermally stable (if in fact it is so)? These two points are the starting points for our investigations. As is the general rule in science, other questions remain to be answered or were generated by previous experiments, some of which we outline below. For this portion of the semester there will be no lectures or protocols provided by the instructional staff for this project. Rather, you will be expected to look at the work done in previous classes, find a topic that you would like to investigate (or design your own novel experiment), conduct a survey of and read the scientific literature for alkaline phosphatase (that can be obtained from Pubmed) that is specific to the experiment you choose to do. Finally, carefully design an experiment in order to answer the question, which will be presented to the instructional staff as a proposal. Timeline :
Remember, it will
be the responsibility of each group to become familiar with the experimental techniques for
their project and design the experiment they will perform, with consultation with Dr. Hazzard,
the TA’s, and Dr. Chad Park (CD). Some experiments will require you to work in labs in BSW
where that CD instrumentation is located. The fluorescence experiments will
be done in Koffler 520A. If a group chooses to do limited proteolysis and the results look
promising, the MALDI-TOF work will be done at the Mass Spectrometry Facility in Old
Chemistry. You are NOT limited to these choices listed below and are FREE and ENCOURAGED to ask you own questions (within reason).Since fall 2009 we have followed the fluorescent and CD spectral changes occuring during thermal denaturation of E. coli alkaline phosphatase, looking at the effect of the reducing agent TCEP on the extent and rate of protein denaturation in either the oxidized or reduced state. During the spring 2011 semester, students from both the morning and afternoon classes examined the effect of anions on the denaturation process followed by CD spectroscopy. In essence, they found that certain anions stablize the enzymes structure while other anions have no stablizing effect whatsoever, in agreement the general precepts of the Hofmeister series. Whether using fluorescence or CD spectrocopies, thermal denaturation experiments are difficult to perform because of the extreme thermal stability of the enzyme. Therefore, this semester we are switching to chemical denaturation of AP using urea and/or guanidinium hydrocholoride (GdnHC). As in the past, one can perform these experiments in the presence of specific cations or anions, in order to test the effects of these ions on the stability of the enzyme. Also, again, we want to perform these experiments with the oxidized (RSSR) and reduced (RSH) forms of the enzyme using TCEP as a disulfide bond reducing agent. 1. Fluorescence spectroscopy is a very sensitive way to monitor conformational changes in proteins during denaturation. Tryptophan is a very good fluorophore to monitor changes in protein structure during denaturation due to the fact that the emission maximum undergoes a significant shift to longer wavelengths as the Trp goes from the non-polar protein interior to the aqueous solvent environment upon denaturation (see the poster in class). Data obtained by previous classes clearly shows that there is a dramatic effect of TCEP on the temperature at which the Trp spectral transition occurs. One complication in these studies was that excitation of Trp fluorophore was done at 280 nm, which results in excitation of Tyrosine residues as well. It would be preferable to repeat these experiments using an excitation at 295 nm which eliminates the complication due to Tyr. It should be remembered, that you should also determine the effect of the denaturants on the fluorescence of free Trp and Tyr. 2. As a corollary to (2) is the use of another sensitive fluorophore, ANS (8-anilino-1- napthalenesulfonic acid), which undergoes a significant shift in its emission spectrum when it binds to hydrophobic portions of proteins during denaturation studies. Last year we attempted to follow thermal denaturation in the presence of ANS which proved to be very problematic due to the effect of heat on the fluorophore itself. Using a heat block, how could you design an experiment where the protein but NOT the ANS is heated? The question to be answered by this experiment is whether or not increased emission due to bound ANS can be observed with protein denaturation? 3. A recent paper has suggested that the two disulfide bonds within AP play two distinct roles.
One disulfide bond is related to enzymatic activity while the other is structural in nature.
Supposedly, AP is resistant to tryptic digestion with the structural disulfide bond intact. In
order to test this hypothesis, one can investigate the digestion of AP by trypsin over a period of
time in the absence and presence of disulfide reducing agents such as BME, DTT, or TCEP.
This type of experiment is generically referred to as a limited proteolysis study. We also need
to carefully consider the temperature at which these experiments are done. There are two
ways to analyze the data: SDS-PAGE and/or MALDI-TOF of the digests. This is a very complex experiment to perform, therefore each group will be limited to looking at limited proteolysis using only ONE reducing agent. This experiment will also require that you take time points spanning a 24 hour time period! You are cautioned to limit yourself to only one or two reducing agents, in addition to looking at the oxidized protein - this is not a trivial experiment to do well. 4. Enzymatic activity of AP is dependent upon the three metal ions. How is enzymatic activity effected by chelating agents such as EDTA? What other common chelating agents are used to bind metal ions, specifically Zn^2+ ions? What are the concentration dependencies for these chelating agents? 5. We have evidence that the thermal stability of AP in the cell lysate, as well in the final Stage 4 enzyme, is greater than for the commercially available, and highly purified, enzyme purchased from Sigma Chemical Company. Note that Garen and Levinthal made the same observation in their paper, attributing the stability to Mg^2+ ions. Based on the tandem mass spec experiments performed in fall 2009, we have evidence that in the Stage 4 enzyme are a number of potential candidates that could help stabilize AP in a chaperonin-like manner. In order to test this, thermal stability assays (doing enzyme activity assays with enzyme held at elevated temperature) could be performed. Obviously, in order to identify potential candidates, in would be desirable to do tandem mass spec experiments on selected "stages" of the enzyme during its purification. 6. In the fall 2009 semester, a group of students did a tandem mass spec experiment on their stage 4 enzyme, getting some very interesting results (to be posted on the power point page). In essence, in addition to AP, several other proteins were detected in a tryptic digestion of the stage 4 enzyme solution. It would be worthwhile to go back and repeat this experiment, however also do a tandem mass spec analysis on the band you believe to be AP from a SDS-PAGE gel. The question for this latter sample being, is the enzyme "pure" in the gel? The sky is the limit on other experiments you might want to perform. Looking at the work done during fall 2010 gives you an idea of different projects you might want to undertake. Remember, it is also possible to repeat what has already been done in order to verify, or refute, another group's data. Remember, you can do searches on PubMed or any other online publication searching site. The following is a partial list of papers that will assist you in developing your experiments. You are also strongly encouraged to use the Internet freely, especially PubMed. Some papers that might help: General Information: Swapna's Data Fitting Lecture: Data_fitting_swapna.pdf Andy Hausrath's Bioc 555 Lecture Notes on Unfolding: Bioc 555_lecture.pdf CD instructions: cd_bundle.doc and calculating molar ellipticity: meanresidueellipticity.pdf Fluorimeter instructions: Quick Guide PTI.doc Protein Unfolding Papers: solvent_denaturation_paceshaw.pdf greenfield_solventdenaturation.pdf Limited Proteolysis Papers: partialproteolysis_levdikov.pdf Transport across cytoplasmic membrane: Processing_AP.pdf Formation of Disulfide bonds in periplasm: DsbB-DsbA.pdf A recent review of the AP mechanism by Herschlag's lab (2008): Comparative enzymology of AP Sulfatase activity of an AP: AP_sulfatase_activity.pdf
R.H. Pain (2004) Current Protocols in Protein Science, Supp. 38, 7.7.1 - 7.7.20. Determining Fluorescence Spectrum of a Protein. S.M. Kelly and N.C. Price (2006) Current Protocols in Protein Science, Supp. 46, 20.10.1 - 20.10.18. Circular Dichroism to Study Protein Interactions. R.H. Pain (2004) Current Protocols in Protein Science, Supp. 38, 7.6.1 - 7.6.24. Determining the CD Spectrum of a Protein. A. Hawe, M. Sutter, and W. Jiskoot (2007) Pharm. Research 25, 1487 - 1499. Extrinsic Fluorescent Dyes as Tools for Protein Characterization. M. Sone, S. Kishigami, T. Yoshihisa, and K. Ito (1997) J. Biol. Chem. 272, 6174 - 6178. Roles of Disulfide Bonds in Bacterial Alkaline Phosphatase. S.N. Sarkar and N. Ghosh (1996) Arch. Biochem. Biophys. 330, 174-180. Reversible Unfolding of Escherichia coli Alkaline Phosphatase: Active Site Can Be Reconstituted by a Number of Pathways. M. Bortolato, F. Besson, and B. Roux (1999) Proteins 37, 310 - 318. Role of Metal Ions on the Secondary and Quaternary Structure of Alkaline Phosphatase from Bovine Intestinal Mucosa. B. Asgeirsson and K. Guojonsdottir (2005) Biochim. Biophys. Acta 1764, 190 - 198. Reversible Inactivation of Alkaline Phosphatase from Atlantic Cod in Urea. J.V. Mersol, D.G. Steel, and A. Gafni (1993) Biophys. Chem. 48, 281 - 291. Detection of Intermediate Protein Conformations by Room Temperature Tryptophan Phosphorescence Spectroscopy During Denaturation of Escherichia coli Alkaline Phosphatase. For those contemplating limited proteolysis, well will be using Trypsin Gold sold by Promega. You can go to their website and look at information on the protease in a pdf format. Biochemistry 463a |
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