Translation of Chemical Biology Research into the Biochemistry

Chapter 10

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Translation of Chemical Biology Research into the Biochemistry Laboratory: Chemical Modification of Proteins by Diethylpyrocarbonate Laura M. Hunsicker-Wang1 and Mary E. Konkle*,2 1Department

of Chemistry, Trinity University, San Antonio, Texas 78212, United States 2Department of Chemistry, Ball State University, Muncie, Indiana 47306, United States *E-mail: [email protected].

Chemical modification of proteins is an ideal system to use to integrate research and teaching. A common chemical modifier, diethyl pyrocarbonate (DEPC), was used to probe the reactivity of the ligating histidine residues in the Rieske protein toward small molecules in a research laboratory. The DEPC work in the research lab inspired a new teaching lab using DEPC to monitor modification of lactate dehydrogenase to teach students to use spectroscopic measurements, structural analysis, and modeling to predict where modifications would be found on the protein. In the original studies in the research lab, saturating amounts of DEPC were added to the samples, and so collection of new data by Biochemistry lab students using less than saturating amounts was used to inform the research lab. The results from the teaching lab led to a several new projects in the research lab that resulted in a publication. These examples demonstrate the usefulness of chemical modification in the research lab, the teaching lab, and the integration of of research and teaching.

© 2018 American Chemical Society Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Introduction


Today’s professor at a primarily undergraduate institution (PUI) has to maintain the precarious balance between research and teaching for both their own personal career development as well as for the benefit of their students. One way to alleviate this tension is to bring research projects into the teaching laboratory. Subsequently, one can use the results from the teaching lab to inform the next steps in the research laboratory. We present a case study of how chemical modification in the research laboratory presented numerous opportunities, both in pedagogical content and additional novel research discoveries (Figure 1).

Figure 1. Overall workflow.

Background One way to determine the functional impact of a particular amino acid in a protein is by methodically changing the amino acid identity at the genetic level through site-directed mutagenesis. However, the expense of the materials and equipment needed for this technique often make it prohibitive for the typical undergraduate biochemistry laboratory course. An alternative and complementary technique to introduce the effect(s) of changing the structure on protein function in the Biochemistry Laboratory is chemical modification. Chemical modification has the benefit of expanding the available organic functional groups past those encoded by DNA. Additionally, it provides a welcome pedagogical link between material taught in organic chemistry courses and its applications to the biochemistry laboratory. In contrast to the reactions carried out in an organic chemistry laboratory, the introduction of a macromolecule such as a protein introduces heterogeneity of reactivity that is both vexing in characterizing the products and rich in teaching opportunities. The reactivity of amino acid side chains in a protein can be influenced by a number of biophysical characteristics such as solvent accessibility, hydrogen bonding, and local pKa values. 166 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Diethyl pyrocarbonate (DEPC) is a small molecule (Figure 2) that readily reacts with free amines in a pH-dependent manner and readily degrades in water to ethanol and carbon dioxide, two molecules that are relatively innocuous to biomolecules. Because many buffers used in the Biochemistry Laboratory contain a free amine, like tris(hydroxymethyl)aminomethane (Tris), care needs to be taken to find an inorganic molecule or non-nitrogen containing organic molecule which can stabilize at the desired pH but will not interfere with the experiment. This allows for an excellent teachable moment about characteristics of a buffer and the importance of knowing its chemical structure in addition to its common name and pKa.

Figure 2. DEPC molecule and the histidine adduct in the Rieske protein.

DEPC is most reactive towards the amino acids lysine and histidine (in the deprotonated state), but also reacts with cysteine and tyrosine residues. The lysine-DEPC adduct is irreversible in contrast to the carboethoxylated histidine-DEPC adduct which is reversible under high pH conditions or upon exposure to hydroxylamine (1). The histidine-DEPC adduct has the advantage of being a chromophore that absorbs at λ = 240-250 nm (ε = 3200 M-1cm-1). Since DEPC-reactive residues are often key in the mechanism of enzymatic activity, DEPC is used as both a probe for active sites that have no structural information available and/or as a deactivator (e.g. DEPC treatment of water is used to deactivate RNAse). The sites of modification can be identified by proteomic analyses using tryptic digestion and LC-MS/MS analysis in an analogous fashion to identification of post-translational modification (2–9). 167 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Research Genesis Complex III in the respiratory electron transport chain accomplishes the oxidation of quinol and transfer of electrons to cytochrome c (10). One subunit of Complex III is the Rieske protein (RP), which contains a [2Fe-2S] cluster that is ligated by two cysteine and two histidine residues (11, 12). RPs couple electron transfer with proton movement across the membrane and thus have pH-dependent reduction potentials. These potentials can be as high as ~ +475 mV at low pH. Thermodynamic characterization of the Thermus thermophilus Rieske protein (TtRp) indicates a low-pH reduction potential of +161 mV with pKox1, pKox2, and pKred values of 7.85, 9.65, and 12.5, respectively (13). When RP is reduced, the electron is localized to the Fe atom bound to the histidine (10). At the time of the original experimental design, the molecular determinant(s) of the pH-dependence was unclear. The Hunsicker-Wang Laboratory, with Dr. Konkle as a postdoctoral researcher, decided to use DEPC as a probe for deprotonated histidine residues that also ligated the [2Fe-2S] cluster (Figure 2).

Original Research Outcomes A truncated form of RP from Thermus thermophilus (truncTtRP) and a mutant that removes the histidines that are not coordinated to the cluster, were modified at a variety of pH values (6.0, 7.0, 8.0) and the modification was monitored with both UV-visible and circular dichroism spectrophotometry over time. The accumulation of the His-DEPC chromophore was observed at λ= 250 nm. Additionally, the impact of modification on the environment local to the [2Fe-2S] cluster can be monitored by ligand to metal charge transfer (LMCT) bands (Figure 3). The DEPC-His chromophore signal (Figure 3b) does not discriminate between the ligating and non-ligating histidine residue. However, the LMCT bands report solely on modification of ligating residue(s) (Figure 3a) (5, 6).

Implementation into the Teaching Lab There were several factors that made translating this experiment into the Biochemistry Teaching Lab feasible. Spectroscopy is a satisfying module in the biochemistry teaching laboratory because results are evident quickly, and numerous companies supply accessible modules for UV-visible spectroscopy in the teaching laboratory. DEPC is an inexpensive and readily available reagent from several commercial sources. The side-reactions (mostly with water) result in the generation of carbon dioxide gas and ethanol, both of which are non-toxic. From an enzymology perspective, many enzymes contain an active site histidine or lysine residue. Therefore, DEPC can be used to illustrate the impact of chemical modification on enzymatic activity and to reinforce the concept of enzyme mechanism taught in a lecture setting. 168 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Figure 3. a) Difference spectra of truncTtRP modified by DEPC and monitored by UV-visible spectroscopy. The arrows denote the DEPC-His chromophore and LMCT band. The raw data is seen in the inset. b) The rate of modification at pH 6.0 (triangles), 7.0 (circles), 8.0 (squares). Error bars shown are the S.E.M. of n=3 and the dashed lines are shown for illustration only. Adapted with permission from ref. (6). Copyright 2010 ACS.

Implementation Implementation 1: Modification of Lactate Dehydrogenase by DEPC Lactate dehydrogenase (LDH) is an enzyme of recent interest as a serum biomarker in cancer and HIV as well as a target for treating diabetes (14–17). There are numerous structures of LDH isozymes from a variety of tissues. The robust and well-documented spectrophotometric assay for LDH activity 169 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


of monitoring NADH production at λ = 340 nm makes it an ideal model system by which to examine the impact of DEPC modification on enzyme structure/function. The experiment consisted of two sections; pH dependence of chemical modification and enzymatic activity assay. The chemical modification (Week 1) and enzyme activity assays (Week 2) was done in groups of two-three students (Figure 4).

Figure 4. Workflow for determining the impact of DEPC modification on LDH enzymatic activity.

LDH Chemical Modification Each group made a buffer of either pH 6.0, 7.0, 8.0, and 9.0 to dilute a control sample of an aliquot of LDH or a matched sample to be modified by DEPC (Sigma Aldrich). The students were responsible for the calculations and pH stabilization of their assigned buffers. In our case, the LDH was purified from a beef heart as part of previous lab exercises, but it is also commercially available. The reaction was begun by the addition of 5 μL (neat) of DEPC to a microcuvette (Starna) containing LDH diluted into the appropriate buffer (375 μL total volume) and was monitored using an Agilent 8453 Spectrophotometer. The spectrophotometer was used to measure the absorbance at 280 nm (to monitor intrinsic protein absorbance of tryptophan, phenylalanine, tyrosine residues) and 250 nm (to monitor the accumulation of the histidine-DEPC adduct chromophore) every 15 s in Kinetics mode after the addition of DEPC. The appropriate buffer was used as 170 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


a blank. Each sample (both treated and untreated) was simultaneously dialyzed using separate labeled dialysis cassettes (Thermo Fisher Scientific) (eight total samples with control and modified samples at four different pH values) into one 2 x 4 L volume of 100 mM phosphate buffer at pH 8.0 at 4° C over 2 x 2 hour periods to remove ethanol from the samples. Due to the time constraints of the laboratory period, the instructor may need to change the dialysis buffer at the appropriate time. The samples were stored at -80°C for one week. For the data analysis for Week 1, the students must plot spectra for individual time points and contribute data to a shared plot of ΔAbs250 vs. time (Figure 5a).

Figure 5. Representative data from the DEPC modification of LDH in the teaching laboratory a) Kinetics of His-DEPC adduct accumulation at pH 6 (triangles), pH 7 (squares), pH 8 (triangles) b) Impact of DEPC modification at pH 6 (light gray), pH 7 (dark gray), pH 8 (open) on enzymatic rate of LDH. Errors shown are S.E.M. 171 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

LDH Activity Assay


Briefly, the students were asked to titrate the amount of control LDH sample needed to achieve a rate between 0.2 – 0.4 ΔAU/min at λ = 340 nm (to monitor NADH degradation) upon the addition of pyruvate and NADH. All kinetic assays were done at 37° C in microcuvettes (375 μL total volume used) on an Agilent 8453 Spectrophotometer in Kinetics mode. Students assayed the modified samples using the same volume of LDH enzyme for comparison. The data analysis for Week 2 was that students must contribute data to a shared plot of activity vs. modification pH (Figure 5b).

Implementation 2: Molecular Modeling of Modification The sites of modification in truncTtRp by DEPC were analyzed using mass spectrometry (6). The ligating residue His154 was identified as being modified. Additionally, the sites of modification not accessible by spectroscopy (mostly lysine residues in this case) were also identified (Figure 6). One lysine residue in truncTtRP, Lys95, was not observed as modified. Lys95 was observed to be in either hydrogen-bonding or ion-pairing interaction with a glutamate residue in crystal structures of TtRP. Since crystal structures of LDH are available, students can be asked to predict the reactivity of histidine and lysine residues based on solvent accessibility and existing molecular interactions using free molecular modeling software (e.g. UCSF Chimera Package). The concept of solvent accessibility was difficult for students to infer from the crystal structures as only 20% of students in a representative lab section had a completely correct answer, 60% had a partially correct answer and 20% received no credit. This study presents an opportunity to improve upon pedagogy and consider additional tools beyond static crystal structures. The students were also asked to identify catalytic residues that would be vulnerable to DEPC modification. In contrast to the solvent accessibility question, 80% of the students answered this question correctly. In summary, the molecular modeling module was completed outside of class and familiarizes students with modeling software, requires critical analysis of lab results, and reinforces the structure/function paradigm.

Implementation 3: Refining Original Research Experimental Conditions The Rieske protein was a wonderful target for DEPC. There are strong spectroscopic changes that can be observed upon modification and therefore could be monitored using techniques that are already taught in a Biochemistry laboratory. All of the original studies were performed using exceedingly high amounts of DEPC (10 µL of neat DEPC into a 300 µL sample) and thus a laboratory experiment was designed for the Biochemistry lab to test what would happen if less DEPC was used. Students were divided into groups and assigned a set of conditions to test the DEPC reaction. They were given one of 2 different pH values and either 2.5 or 5 µL of DEPC added. Students then collected both 172 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


UV-Visible and CD spectra, and the groups shared their data to develop a profile of the effect of pH. Ultimately, what the students and the instructor learned was that even decreasing the DEPC amounts by ½ to ¼, the resultant spectra are identical. Therefore, in order to fully probe the effect of different amounts of DEPC, a method to achieve lower concentrations was needed. Fresh dilutions of the near DEPC into 200 proof ethanol proved to be the way to attain the lower number of equivalents. This method was then applied to the wild type protein and the H120Q/H162Q mutant in the research lab. Thus, the full study was supported by work carried out in the Biochemistry teaching lab (7).

Figure 6. Modification of truncTtRP identified using proteomic analysis. a) Crystal structure of TtRP illustrating the residues modified by DEPC b) Table describing the modified residues of TtRP in various forms. Adapted with permission from ref. (6). Copyright 2010 ACS.

Analyzing Modified Protein Using Isoelectric Focusing Gels, UV-Visible and CD Spectroscopies Chemical modification of proteins alters many properties of the proteins, and thus several analytical methods are available to characterize the modified protein. It is powerful to use these techniques in the context of a complex reaction rather than in a “canned lab”. The techniques that were utilized were UV-Visible and CD spectroscopies and Isoelectric Focusing gels. UV-Visible spectroscopy was used to monitor the formation of the DEPC adduct since there is a chromophore that forms upon reaction with the histidine (1). CD spectroscopy was used to monitor the changes at the [2Fe-2S] cluster, including the reduction that occurs following modification by DEPC (7). The students each monitored the two different reactions using both techniques and 173 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


could compare the effect of either pH or the different amounts of DEPC. For many students, CD is not a technique that they have typically used and if they have, it is usually monitoring the low wavelength regions for changes in secondary structure. Thus, the laboratory gives them an opportunity to use CD in a different way and to explore its capabilities in the context of chemical modification. It is also powerful for the students to compare the UV-Visible and CD results since the two techniques report on different aspects of the modification. The UV reports on the histidine-DEPC adduct formation, whereas the majority of the spectral change in the CD derive from the reduction of the cluster. Examining how these correlate leads to a deeper appreciation of the complexity of the reactions. Isoelectric focusing (IEF) gels were also utilized to characterize the products of the chemical modification. DEPC reacts with the lysines and the N-terminus which will lower the isoelectric point (pI) of the protein, resulting in a change that can be visualized using IEF gels. The students saved their CD samples after modification (stored at -80 C), and then loaded 5 µL of the reaction mixture onto the IEF gel. They then compared those samples to unmodified protein and to a ladder to determine the pI of the unmodified protein and how the pI changed. They could also determine if all of the protein sample was modified by noting if any protein with the pI of the unmodified protein was present on the gel. This exercise was especially helpful for students to appreciate the amount of modification that was taking place, as nearly all of the sample changed pI. As a multi-week lab, students were assigned one of two weeks to come to the lab and conduct the UV and CD experiments. The data from all of the students was shared so that the students could compare their data to others in the lab. Then in the final week, all the students were in the lab together running the IEF gels. Once all the experiments were completed, the students wrote up the report using their CD and UV data, the shared CD and UV data and their IEF gels.

Implementation 4: Extending to Other Proteins in Research and Teaching The CuA protein has also served as a great target for DEPC modification. The CuA protein is subunit II of cytochrome oxidase (Complex IV) of the ETC. It contains a dinuclear copper cluster where the two copper ions are bridged by two cysteines, each copper is ligated by one histidine, and a methionine and a glutamine carbonyl from the backbone complete the ligation environment (Figure 7) (18). Given its structural similarity to the Rieske protein, it is a good candidate for reactivity with DEPC. The protein was therefore subjected to reaction with 400 eq of DEPC at pH values 5-9. The reaction was again monitored using UV-visible and CD spectroscopies. The reaction is more extensive under higher pH reaction conditions which is consistent with a reactive histidine residue. A mutant, which removes the non-ligating histidine residues, H40A/H117A, was also tested. There was still modification when there were the only ligating histidine residues. Thus, all the data points to a ligating histidine being modified (19). In the research lab, all of these studies used 400 equivalents of DEPC, which is at the solubility limit of DEPC. 174 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.


Figure 7. a) Structure of the ba3 cytochrome c oxidase from Thermus thermophilus (1XME). b) The CuA metal site with ligands and the important Asp 111 shown.

In order to take this project into the teaching lab, a laboratory protocol was developed in order to test what would happen when the protein was subjected to 1-200 equivalents of DEPC. Different groups were assigned lower values of DEPC. The teaching lab students discovered that the spectroscopic changes that occur in the CD spectrum happen with as few as 3 equivalents of DEPC! Thus, a very small amount of DEPC is needed to observe changes at the copper cluster. This result was completely unexpected, considering that for the Rieske protein, very little change in the spectrum was observed with 1-6 equivalents (7). Like the Rieske lab, students also analyzed their data using IEF gels and noted that the pI decreased with modification but with the lower amount of DEPC added, some unmodified protein remained. These results have led to further studies in the research lab which corroborate the results of the teaching lab and are being incorporated into an upcoming publication (19). In the biochemistry lab, each experiment is graded by students turning in a report that consists of results and discussion sections that are modeled off of manuscripts. Students present the data that they collect in the results section and then are asked to put their data in context in the discussion section. The discussion section is facilitated by the students answering a series of discussion questions. The answers to the questions are all written in paragraph form. These types of questions necessitated students looking beyond their own data and also forced them to use multiple types of data to understand what is happening in the reaction. 175 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Assessment Discussion Questions from the CuA Chemical Modification (Similar Questions Were Asked about Rieske) A file with all the groups’ sets of UV and CD data was compiled and a plot of 240 vs. the number of equivalents was produced and included in the PowerPoint file. The file was put on a learning management system and the entire section had access to it. Answer the following questions looking at the data from across the section


1.

2.

3.

Looking at the A240 nm data from the UV allows us to determine the rates of modification (from A240 nm) since the 240 nm absorption is due to the histidine-DEPC adduct. Looking at the increase in equivalents across the section, what trends do you see in the rates of modification? Looking at the 440 nm data from the CD allows us to look at the changes localized at the cluster due to the modification. This statement is true because all of the changes in ellipticity in the visible region of the CD originate from the Cu2S2 cluster. Looking at the increase in equivalents across the section, what trends do you see in the changes at the cluster? Do the changes in the UV and CD correlate?

One desired student learning outcome for the lab would be a deep understanding of the techniques that are taught in the lab. To this end, the chemical modification should help students to better grasp the techniques that are used to analyze the products of the chemical modification. CD, UV-Vis and IEF gels are used for the analysis and the students that perform the chemical modification should perform better on an extension question on a final exam that asks them to explain a phenomenon that they had observed, but that we had not discussed directly. In two different semesters of the biochemistry teaching lab, students were asked the same question (with a slight change to wording to be appropriate for the given lab) about IEF gels on the final exam. One semester we used the IEF gel to help analyze the results of the chemical modification. In the other semester, the IEF gel was used to identify an unknown protein from a set of possibilities, using pI as the determinate factor. The question was worth 5 points and is given below. In the year when chemical modification was used, the average score was 3.6 out of 5, where the average answer from the year chemical modification was not used was 3.2. Thus, there is preliminary evidence that the use of chemical modification may help students better understand IEF gels. It will be interesting to compare more years of exams to see if this is a reproducible trend.

176 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

Final exam questions: Semester without chemical modification (n= 18) You all ran pI gels of several proteins. If you looked carefully at the isoelectric focusing gel while it was running, you could actually see the brown bands of the heme-containing Myoglobin progressing through the gel. If you ran an SDS-PAGE gel of the same sample at the same concentrations, you would not be able to see those same brown bands progressing through the gel. Explain why using 1-3 sentences.


Semester with chemical modification of the Cu A protein (n=10) If you looked carefully at the isoelectric focusing gel while it was running, you could actually see the purple bands progressing through the gel. If you ran an SDS-PAGE gel of the same sample at the same concentrations, you would not be able to see those same purple bands progressing through the gel. Explain why using 1-3 sentences. A second way to assess student engagement is to look at course evaluations. The following is an example quotation from the course evaluation of laboratory course when the chemical modification of Rieske was used. “I really liked the rieske lab. I think labs are far to guided at all levels with results able to be calculated before the lab is even done. I think more labs should be open ended and working without a net because that’s how it happens in "the real world". A second assessment of the student reception of the material is through a question on the final exam. The last exam question is always “What was your favorite lab and what was your least favorite lab this semester and why? (There is no wrong answer but you must answer which one AND why).” In the year when we did the chemical modification of CuA, 50% of the class answered that the chemical modification was their favorite lab or tied for their favorite lab. For perspective, the following year when chemical modification was not in the lab, the “favorite lab” was chosen by 28% of the class. Thus, a higher percentage of students chose the chemical modification lab as their “favorite” one.

Ideas for Extensions and Limitations There are several ways to implement or extend the given examples directly. One could envision modifying other commercially available proteins that would have critical histidine residues, such as carbonic anhydrase, RNAse, DNAse, etc. The limitations here are that the proteins need to have solvent accessible histidine residues to detect a spectroscopic change or a relatively open active site to detect changes in enzymatic activity after modification. For example, cytochrome c has an active site histidine, but it is not accessible and DEPC has been shown to only react with the surface histidine residues (4, 20). Perhaps more significantly, the authors believe that this can serve as an example of how to integrate the research and teaching purposes for both faculty and students. 177 Gourley and Jones; Best Practices for Supporting and Expanding Undergraduate Research in Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 2018.

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Translation of Chemical Biology Research into the Biochemistry

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