Implementing HSQC-NMR a New Tool for Directed Evolution of Myoglobin with Kemp Eliminase Properties
Discipline: Chemistry and Chemical Sciences
Subcategory: Cell and Molecular Biology
Session: 3
Sonia Lizzette Ortiz - Laredo College
Co-Author(s): Jennifer Yoon, Syracuse University, NY; Sagar Bhattacharya, Syracuse University, NY; Olga V. Makhlynets, Syracuse University, NY
Proteins can be evolved to accept unnatural substrates or to catalyze new chemical reactions. The process of evolution is guided by available structural information or by computational analysis. A new method for the evolution of enzymes was developed; which relies on the differences in HSQC spectra of the protein in the presence and absence of substrate analog. This method has been successfully used to improve catalytic efficiency of calmodulin-based Kemp eliminase that operates through the acid-base mechanism. The goal of this work is to demonstrate an effective approach to apply other protein scaffolds and reaction mechanisms. Recent work by the Reetz group shows that Kemp elimination is promoted by coordination of Kemp substrate to the Fe(II)-heme center of cytochrome P450-BM3. Subsequent electron transfers between iron and Kemp also occur. To test the applicability of NMR guided evolution to redox-catalyzed Kemp elimination; a project with heme-containing protein myoglobin (Mb) was conducted. An H64V Mb mutant protein and NMR analysis were performed to gain an insight into the interactions between substrate and protein for catalysis. Unlabeled Protein: Mutation H64V, pET-28a vector was introduced to myoglobin previously before entering the lab. A transformation took place and colonies were selected for growth. Proteins grew in 1 XLB (starter culture) at 37°C for 6 hours. As a result, it becomes cloudy. During the waiting period, 2 liters of TB culture media and 1 liter of unlabeled M9 media were formulated. Two cell lines grew proteins, BL21 (DE3) and BL21 plysS. OD600 (Optical Density) reached 0.8, which was diluted with the 2 liters of TB media and placed back into the shaker and continued to check OD. The solution was spun down at 4,000g for 30 minutes, which gave cell pastes in BL21 DE3 and BL21 plysS. Poured out the solution and washed the pastes with 15mL of unlabeled M9 media and centrifuged it at 4,000g for 30 minutes. Cell paste was transferred to 1 liter of M9 media and induction was conducted. We grew protein again at 25°C for 20 hours and spun it down at 4,000g for 30 minutes getting paste 1 and 2. Purified our proteins and found the final concentrations of both BL21 DE3 (600µM) and BL21 plysS (517µM). With these results, we decided to grow double labeled protein using BL21 DE3. Labeled Protein: Following the previous process, 2 liters of labeled M9 media were produced. Protein was induced with 0.3mM of aminolevulinic acid (heme precursor) and 0.25mM of IPTG placed in shaker at 25°C for 20 hours. Overnight growth and centrifuged at 20,000g for 30 minutes occurred; cell pellets were collected and continued with resuspension using 25mM of TRIS buffer at pH 8.0. We then sonicated having 20 seconds for pulse, and 20 seconds for rest in total of 10 minutes. Centrifuge at 20,000g for 30 minutes disposed of the cell debris and combined both fractions of protein. Increased concentration of the protein and load to Ni-NTA column. We used 25mM TRIS buffer at pH 8.0, and 20mM imidazole increased the amount of imidazole to 50mM and 250mM. Using a desalting column, we looped 3mL buffer exchange of 20mM of HEPES pH 7.0 and lastly store in 20 mM HEPES buffer at pH 7.0. During these studies, we successfully introduced H64V mutagenesis into Mb and observed the shifts of it using HSQC spectra. We are in the process of determining what from the spectra due to the movements of the Mb. In the use of HSQC spectra, we’ve identified changes of H64V Mb purified that include His tags and one without, along with 15N-labeled Mb protein which was compared between reduced ad oxidized forms of iron-bound Heme Mb. A substrate analog to the H64V Mb shifts some of the peaks in the spectra, suggesting that the substrate is interacting with the corresponding residues. In this work, we successfully cloned H64V Mb into pET-28a and established a reproducible Mb protein expression protocol to prepare [13C, 15N] – labeled Mb for HSQC-NMR spectral analysis. Further work, will focus on the expression of [13C, 15N] – labeled Mb and assignment of peaks observed in HSQC. After this, we will titrate the protein with the inhibitor and identify which residues shift. Based on NMR analysis we will introduce mutations in the vicinity of heme and screen for accelerated formation of a yellow 2-cyano-4-nitrophenol product. Our work presents the validation of a novel method developed for the design of artificial enzymes.
Funder Acknowledgement(s): I would like to thank the National Science Foundation (CHE 1659775) The Upstate Louis Stokes Alliance for Minority Participation (LSAMP) program and Syracuse University for funding and allowed to participate in the summer REU program this summer. I would also like to personally thank Dr. Olga Makhlynets and Dr. Ivan Korendovych for allowing me to work on the project and in their lab. Jennifer Yoon and Sagar Bhattacharya for sharing the best of their knowledge, making time to explain the project and guiding me throughout my time in and out of the lab, and both Makhlynets and Korendovych lab.
Faculty Advisor: Olga V. Makhlynets, ovmakhly@syr.edu
Role: During the research, I assisted in making starter culture buffer and creating plates for the growing our cells. In addition, helped with growing our cultures and purify them for testing.