SJSU Annual Program Assessment Form Academic Year

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1 SJSU Annual Program Assessment Form Academic Year Department: Chemistry Program: MA/MS College: Science Program Website: Link to Program Learning Outcomes (PLOs) on program website: Program Accreditation (if any): None Contact Person and Karen Singmaster, Department Chair, Annalise Van Wyngarden, Interim Graduate Committee Chair, Date of Report: June 1, 2016 Part A 1. List of Program Learning Outcomes (PLOs): See Table 1 below. 2. Map of PLOs to University Learning Goals (ULGs) : See Table 1 below. 3. Alignment Matrix of PLOs to Courses: See Table 1 below. Note that the Graduate Program does not offer any specific 200-level lecture courses on a regular basis. Course offerings depend largely on faculty availability, as determined in the preceding semester.

2 TABLE 1: PLO Alignment Map for Chemistry Graduate Program 4. Planning Assessment Schedule TABLE 2: Assessment Schedule for Chemistry Graduate Program Key: C = Collect data; D/R = Discuss and Report; I = Implement recommendations SEMESTER PLO F12 S13 F13 S14 F14 S15 F15 S16 F16 S17 F17 S18 1 C D/R I C D/R 2 C D/R I 3 C D/R I 4 C D/R I 5 C D/R I Notes: Data related to all 5 PLOs will be collected continuously from each student that presents a Preliminary or Final Seminar or submits a thesis, in addition to any data obtained from 200- level lecture courses. Fill indicates completed activity, no changes to schedule. 5. Student Experience PLOs are communicated to students via course greensheets and the department website.

3 Part B 6. Assessment Data and Results Summary In this reporting period, data were collected from CHEM 234 Enzymology taught by Professor Rascón in Fall 2015 for PLO #4: To demonstrate a familiarity with available instrumentation for conducting specific scientific research. Specifically, questions from the midterm exam that assessed student achieved of PLO #4 were examined with average scores summarized in Table 3. TABLE 3: Assessment Data for PLO #4 Fall See detailed question descriptions below. Question #1 Question #7 Question #12. b. Question #13. a. Question #13. b. Points Average Score These are averages from 19 students enrolled in the class. Detailed description of assessment: In the Department of Chemistry we have several major instruments available for our Biochemistry lab courses and research labs. These include a Circular Dichroism Spectrometer, an Ultracentrifuge, NMR s (both a Varian 400MHz and a Varian 300 MHz), fluorometers, Thermal cyclers, and UV-VIS spectrophotometers. In addition, the Department has access to core facilities, such as the PROTEIN Lab, which is dedicated to those wishing to conduct proteomics (large-scale study of proteins, their structure and function) or metabolomics (the study of metabolites present in an organism or tissue). The PROTEIN Lab has a variety of instruments such as a Fast Protein Liquid Chromatography (FPLC AKTA Purifier for protein purification), a Quadrupole TOF LC/MS, and a Multi-mode Plate Reader. Our graduate courses are dedicated to introducing the theory behind many of these instruments in order to help students understand the function and possible applications to their own research or even expand on their current knowledge. (Many of our graduate students work in industry or biotech, so this allows them to learn new techniques). In Chem 234: Enzymology we discussed techniques relevant to the study of enzymes. For example, UV- VIS spectrophotometers are used for kinetic assays and following the change in absorbance of specific chromogenic substrates, but students typically forget that these spec units can be coupled to protein purifiers to allow the identification of eluting proteins during the purification process. This is possible because many proteins and enzymes contain amino acids that can absorb UV-light and give off a characteristic absorbance spectrum. Question #1 in the attached Midterm asked about the importance of these aromatic amino acids. Additionally, for better student understanding of how the spectrophotometer is coupled to the FPLC, and how an FPLC works, short answer Question #12b was included. However, rather than asking directly how FPLC works, the question asked to explain why purifying an enzyme from a total mixture leads to a decrease in total activity but an increase in specific

4 activity. A table from a research article was included in the question. The use of Thermal Cyclers is important for Polymerase Chain Reactions (PCR), which allow the ease of amplifying specific DNA genes from low starting materials in order to clone the gene into expression vectors. This allows for large-scale expression, and eventual purification of the protein/enzyme of interest. PCR is a technique that can be used to make alterations in gene sequences, in a process known as site-directed mutagenesis. This was discussed in class in detail, but, again, rather than ask the question directly, the importance of its application was asked relevant to another biochemical technique that uses site-directed mutagenesis. This can be found in Question 13a. As a follow up, Question 13b asked about a specific instrument (Circular Dichroism Spectrometer), which is available for use in the Department. This instrument is used to measure the secondary structure nature of a particular protein/enzyme, so any increases in temperature will lead to denaturing the protein/enzyme leading to disorder and losing the characteristic alpha-helical or beta-sheet content of the protein/enzyme. Lastly, a multiple-choice question (Question 7) asked about the importance of Mass Spectrometry (MS) and macromolecules. MS is an important technique in Biochemistry because with Electron Spray Ionization (ESI) methods proteins and nucleic acids can be studied and used to determine the content/sequence of the given protein/enzyme (in a complex or purified mixture) or DNA/RNA molecules. Students really appreciated the discussion on all of the techniques, especially the MS discussion. Many students enrolled in Chem 234 also took an LC-MS course (Chem 270L, Advanced Chemistry Laboratory), which focused on small molecules rather than proteins (with the same principles applied), but with a hands on approach. 7. Analysis It can be seen from Table #3 that student responses to the midterm questions demonstrated that overall they achieved the PLO assessed (to demonstrate a familiarity with available instrumentation for conducting specific scientific research). Questions #1, 7 and 13b directly assessed this PLO by asking specific questions about instrumentation. For these questions, excellent average scores were achieved. Questions #12b and 13a had lower average scores, most likely since they required more advanced thinking since students not only had to master PLO #4, but also data analysis (#12b) or evaluation of importance of an application to another biochemical technique (#13a). The data for these questions are included here since they demonstrate that many (but not all) students demonstrated achievement beyond the assessed PLO. 8. Proposed changes and goals (if any) Since the assessment showed achievement of PLO #4, there are no recommended changes for this particular PLO.

5 Part C Proposed Changes and Goals Status Update None Discuss the possibility of placing an Advising Hold on registration for all continuing graduate students such that they must meet with the Graduate Advisor and report their status each semester Due to a recent program change that allows more units of research to count toward the MS degree, the Chemistry Department s Graduate Student Handbook should be updated by the start of the F15 semester. The Graduate Committee rejected the Advising Hold and instead suggested various other potentially more efficient and/or effective options for tracking student progress toward their degrees. The decision of which option to pursue is tabled pending further examination by the committee in Completed. Attachments: Chem 234 Midterm Exam Answer Key Sample of a scored student CHEM 234 Midterm Exam

6 Name: CHEM 234 Midterm Exam - Lectures 1-8 (Intro to Enzymology - Enzyme Kinetics: Michaelis-Menten Kinetics, etc ) October 15 th, 2015 Part 1: Ready? And away we go!! (50 pts) For Multiple Choice Questions 1-10, choose the single best answer. 1. Which of the following statements about aromatic amino acids is correct? A. All are strongly hydrophilic. B. Histidine s ring structure results in its being categorized as aromatic or basic, depending on ph. C. On a molar basis, tryptophan absorbs more ultraviolet light than tyrosine, and tyrosine absorbs more UV light than phenylalanine. D. The major contribution to the characteristic absorption of light at 280 nm by proteins is the phenylalanine R group. E. The presence of a ring structure in its R group determines whether or not an amino acid is aromatic. 2. Amino acids are ampholytes because they can function as either a(n): A. acid or a base. B. polar or a nonpolar molecule. C. neutral molecule or an ion. D. standard or a nonstandard monomer in proteins or polypeptides. E. transparent or a light-absorbing compound. 3. A nonapeptide was determined to have the following amino acid composition: (Lys)2, (Gly)2, (Phe)2, His, Leu, Met (shown in no particular order). The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; the product 2,4-dinitrophenylhistidine was then identified by HPLC. When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered. Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys. 2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4- dinitrophenylphenylalanine was recovered from the tripeptide. Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide. The tetrapeptide was composed of (Lys)2, Phe, and Gly. The native sequence was determined to be: A. Gly Phe Lys Lys Gly Leu Met Phe His. B. His Leu Gly Lys Lys Phe Phe Gly Met. C. His Leu Phe Gly Lys Lys Phe Met Gly. D. His Phe Leu Gly Lys Lys Phe Met Gly. E. Met Leu Phe Lys Phe Gly Gly Lys His. Points Possible Points Earned 15 1

7 Name: 4. The most important contribution to the stability of a protein s conformation appears to be the: A. entropy increase from the decrease in ordered water molecules forming a solvent shell around it. B. maximum entropy increase from ionic interactions between the ionized amino acids in a protein. C. sum of free energies of formation of many weak interactions among the hundreds of amino acids in a protein. D. sum of free energies of formation of many weak interactions between its polar amino acids and surrounding water. E. stabilizing effect of hydrogen bonding between the carbonyl group of one peptide bond and the amino group of another. 5. Which of the following pairs of bonds within a peptide backbone show free rotation around both bonds? A. Cα C and N Cα. B. C=O and N C. C. C=O and N Cα. D. N C and Cα C. E. N Cα and N C. 6. Thr and/or Leu residues tend to disrupt an α-helix when they occur next to each other in a protein because: A. an amino acid like Thr is highly hydrophobic. B. covalent interactions may occur between the Thr side chains. C. electrostatic repulsion occurs between the Thr side chains. D. steric hindrance occurs between the bulky Thr side chains. E. the R group of Thr can form a hydrogen bond with Leu. 7. A major advance in the application of mass spectrometry to macromolecules came with the development of techniques to overcome which of the following problem? A. Macromolecules were insoluble in the solvents used in mass spectrometry. B. Mass spectrometric analyses of macromolecules were too complex to interpret. C. Mass spectrometric analysis involved molecules in the gas phase, which destroyed proteins and nucleic acids. D. Most macromolecules could not be purified to the degree required for mass spectrometric analysis. E. The specialized instruments required were prohibitively expensive. Points Possible 20 Points Earned!2

8 Name: 8. Use the following plot to answer the following question: Which of the following statements about this V0 vs. [S] plot for an enzyme that follows Michaelis-Menten kinetics is false? A. As [S] increases, the initial velocity of reaction V0 also increases. B. At very high [S], the velocity curve becomes a horizontal line that intersects the y-axis at Km. C. Km is the [S] at which V0 = 1/2 Vmax. D. The shape of the curve is a hyperbola. E. The y-axis is a rate term with units of mm/min. 9. An enzyme-catalyzed reaction was carried out with the substrate concentration initially a thousand times greater than the Km for that substrate. After 9 minutes, 1% of the substrate had been converted to product, and the amount of product formed in the reaction mixture was 12 µmol. If, in a separate experiment, one-third as much enzyme and twice as much substrate had been combined, how long would it take for the same amount (12 µmol) of product to be formed? A. 1.5 min B min C. 27 min D. 3 min E. 6 min 10. For the simplified representation of an enzyme-catalyzed reaction shown below, the statement ES is in steady-state means that: k1 E + S ES E + P k-1 k2 k-2 A. k2 is very slow. B. k1= k2. C. k1= k-1. D. k1[e][s] = k-1[es] + k2[es]. E. k1[e][s] = k-1[es]. Points Possible 15 Points Earned 3

9 Name: Part 2: Really?!? Come on!! 11. (16 pts.) The Michaelis-Menten equation allows a biochemist to experimentally determine the Km and Vmax kinetic parameters. However, these parameters alone are not enough to compare enzyme activities. Therefore, two other parameters, the turnover number (kcat) and the specificity constant (kcat/km) are used to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme. A perfect example of a journal article using the specificity constant to show how a cofactor/substrate can influence and change the kinetic parameters of an enzyme was that of the Babesia microti lactate dehydrogenase. In the following table, the authors show which kinetic parameters were determined. Using the information in the table as a guide, why is it much more important to use kcat/km together (the specificity constant) to compare the catalytic efficiencies of the lactate dehydrogenase reaction in the presence of NAD + or lactate (heck, even enzymes in general) rather than each parameter alone (kcat, Km, Vmax)? Hint: Think catalytic perfection discussion from class. Explain in detail! Slides Lecture 8: The parameters kcat and Km also allow us to evaluate the kinetic efficiency of enzymes, but either parameter alone is insufficient for this task because enzymes catalyzing different reactions may have the same kcat (turnover number), yet the rates of the uncatalyzed reactions may be different and thus the rate enhancements brought about by the enzymes may differ greatly. In addition, experimentally, the Km for an enzyme tends to be similar to the cellular concentration of its substrate. For example, an enzyme that acts on a substrate present at a very low concentration in the cell usually has a lower Km than an enzyme that acts on a substrate that is more abundant. These values may differ greatly, and only the ratio between them (kcat/km) do we get a catalytic efficient value with a second order rate constant (M -1 *s -1 ), indicating whether the enzyme has reached the diffusion-controlled limit (catalytic perfection) of 10 8 to 10 9 M -1 *s -1. Therefore, the best way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to compare the ratio kcat/km for the two reactions. And from the table, this is obviously observed when either NAD + or Lactate are used as substrates (it is important to note that both are substrates of BmLDH and the only way to compare two different substrates by the same enzyme is through kcat/km). The Km for each is so different (almost 10-fold different), but the Vmax is not as different (only ~1.15-fold) indicating that the enzyme s activity is reaching nearly the same Vmax. This could also be said about kcat, which are nearly close for both substrates. Alone these numbers do not mean much because one could almost say that they have the same catalytic activity, and not until you measure the specificity constant does the enzyme in the presence of the NAD + substrate reach catalytic perfection, while lactate does not. Points Possible 16 Points Earned!4

10 Name: 12. (12 pts.) A trypsin-like protease from the nematode Steinemema carpocapsae (a parasitic nematode) was isolated using various purification techniques. These proteolytic enzymes are responsible for cleaving peptide bonds. a. The hydrolysis of peptide bonds is a highly exergonic reaction. Why, then are peptide bonds quite stable? Explain. (5/12) Peptide bonds are stable because hydrolysis of peptide (or amide) bonds has a high activation energy and as a result occurs very slowly. This high activation energy is due to the peptide bond having double-bond character, there is resonance or partial sharing of two pairs of electrons between the carbonyl oxygen and the amide nitrogen. However, the answer is high activation energy that needs to be overcome and so require proteases to lower the activation energy to hydrolyze the peptide bond. b. The authors of the S. carpocapsae article presented the following table showing the various purification steps used to isolate the trypsin: As shown in the table, as the enzyme is purified, both the amount of total protein and the total activity of the purified protein decrease. Why, then, does the specific activity of the purified protein increase? Explain! (7/12) In this case, the specific activity is the units of enzyme activity (U) divided by the amount of protein (mg). As the enzyme of interest is purified, some of it is lost in each step, resulting in a drop in total activity. However, other contaminating proteins are lost to a much greater extent. Therefore, with each purification step, the purified enzyme of interest constitutes a greater proportion of the total amount of enzyme, resulting in an increase in specific activity. Other contaminant proteins or enzymes that interfered with activity were removed during the purification step. There is also a possibility that in the total protein (crude lysate) there were other enzymes that could contribute to the overall activity, and as the enzyme is purified, the total activity decreases. Points Possible 12 Points Earned 5

11 Name: Part III: Almost THERE!!! Finally!! Jeez 13. (22 pts.) Unnatural amino acids can be used probe the structure and function of protein and enzymes. This approach utilizes many biochemical techniques to make alterations in the gene sequence that encodes the enzyme of interest. To introduce these unnatural amino acids, a process known as nonsense suppression is used. a. Why is site-directed mutagenesis an important part in the nonsense suppression process? Hint: What is nonsense suppression? How does nonsense suppression work? Explain! (12/22) In nonsense suppression, a stop codon (usually the amber codon, UAG) is used to replace a codon that encodes a specific amino acid. This new codon will encode for the stop codon, but rather than end protein translation, the expression system used will have the necessary trna s with the unnatural amino acid that will recognize the amber codon and introduce that unnatural amino acid and allow translation to continue. However, to alter the gene encoding the enzyme of interest, site-directed mutagenesis is used. Using this process will allow one to mutate the WT/native codon to the amber codon allowing recognition of the specific trna with unnatural amino acid. So, during protein translation, rather than terminate protein translation, an unnatural amino acid will be incorporated, as long as their is ample unnatural amino acids in the growth media. It is important to note that the DNA gene of interest is modified by SDM, which when transcribed to mrna, the mrna will have the correct amber codon mutation, then during protein translation the trna s with the unnatural amino acid will be incorporated. b. Introduction of an unnatural amino acid by nonsense suppression can lead to structural changes in the enzyme. For example, a parasitic enzyme functioning in its wild type (WT) state may denature only at higher temperatures (>60 o C). However, the presence of the unnatural amino acid may alter the denaturation temperature (facilitating the melting of the enzyme at temperatures lower than 60 o C ). One way to determine if there is an effect on structure is through the use of circular dichroism. Explain how circular dichroism spectroscopy could be used to measure the denaturation of a protein. (5/22) Circular dichroism spectroscopy measures the amount of α-helix, beta-sheet or random coil in a given protein. As the protein denatures, the amount of α-helix (or beta-sheet) should decrease as the protein chain becomes disordered; this change would be detectable using CD spectroscopy. c. Interestingly, changes in ph can also alter the conformation of an enzyme. How can changes in ph alter the conformation of a protein? Hint: What happens to amino acids when the ph changes? (5/22) Changes in ph can influence the extent to which certain amino acid side chains (or the amino and carboxyl termini) are protonated. The result is a change in net charge on the protein, which can lead to electrostatic attractions or repulsions between different regions of the protein. The final effect is a change in the protein s three-dimensional shape or even complete denaturation. Points Possible 22 Points Earned!6

Purification: Step 1. Lecture 11 Protein and Peptide Chemistry. Cells: Break them open! Crude Extract

Purification: Step 1. Lecture 11 Protein and Peptide Chemistry. Cells: Break them open! Crude Extract Purification: Step 1 Lecture 11 Protein and Peptide Chemistry Cells: Break them open! Crude Extract Total contents of cell Margaret A. Daugherty Fall 2003 Big Problem: Crude extract is not the natural