Assay Conditions Dates to keep in mind 1 Project 3 due the week of March 31/April 1-2 Introduction/figures/future work Individual assignment To be included in the final assignment (manuscript) If you feel that you cannot turn it in this week, we will accept up to April 6 at 1:00 pm (class time), however you risk not getting feedback early enough to incorporate changes into your poster(s) and manuscript (grading turn-around is 1 week) Posters: ACS poster session Friday, April 17th from 6:00-7:00pm April during class times (you will sign up for a presentation time) This is a great opportunity for your undergraduates to present their research and meet with their peers. Last year we had over 20 posters in the session, representing colleges and universities from Tidewater to the Valley. Please do emphasize to your research- oriented undergraduates that they will be most welcome to present their work at this regional meeting of the ACS. More information will be sent out in March or contact Cindy Knight. Bioinformatics Structure and sequence alignments 2 3NRA 1GDE 1J32 1DJU 1BJW 1ASL B A Figure 3 Active site homologue overlays with 3NRA that demonstrate good overlap. (A) Active site of 3NRA and 1J32. (B) Overlays of 3NRA, 1GDE, 1J32, 1DJU, 1BJW, and 1ASL. Good structural overlap suggests similarity in function. 3 POI POI The POI shows 97% sequence homology and highly conserved active site residues with an aspartate aminotransferase from Genus species (GsAspAT). For active site residues, the POI residue is the first number and the GsAspAT is second. The mutagenesis tool was used in PyMOL to remove PLP from modified lysine. 4 Figure 2: Conserved substrate specific and catalytic residues among homologous proteins of LdCBL. Homologous proteins were determined using BLAST sequence and DALI structural alignments with LdCBL. In each PyMOL panel, LdCBL is presented as black cartoons with specific residues highlighted as sticks. Likewise, each homologous protein is presented as blue cartoons and sticks. Residues identified as substrate specific and catalytic residues are shown. 5 Transaminase C StVPAT PaVPAT Transaminase C StVPAT PaVPAT Transaminase C StVPAT PaVPAT Transaminase C StVPAT PaVPAT Figure 2. Sequence alignment of PaVPAT with Transaminase C from E. coli and putative VPAT from S. typhimurium. (3G7Q; StVPAT). Overall identities for PaVPAT relative to Transaminase C and StVPAT are 44.3 % and 44 %, respectively. Strength of alignment demonstrates homology with functionally characterized Transaminase C and structurally characterized StVPAT. 6 Assay and Experimental Design 7 Figure 3. Mechanism of the AspAT/MDH-coupled assay. RsAspAT catalyzes the transamination between aspartate and 2-oxoglutarate, yielding L-glutamate and oxaloacetate. 8 Oxaloacetate then couples with NADH and malate dehydrogenase to produce malate and NAD +. The consumption of NADH is observed. 8 Cloning & Expression Gene for 3NRA was cloned into P BAD vector and transformed into Escherichia coli cells by JCSG 1 Expression was induced with arabinose Purification Immobilized metal ion affinity chromatography (IMAC) protein purification with NiNTA resin Dialysis & Quantification A variety of dialysis buffers were used to optimize the solubility of 3NRA 3NRA concentration was measured via A 280 Progress Curve Optimal concentration of 3NRA was determined to be 2 M Kinetic Assay Concentration of aspartate was varied K M and V MAX values were extrapolated from Michaelis-Menten curve A. Procedural Flow ChartB. Schematic of Coupled AspAT/MDH Assay 9 10 Putting your work into a larger context Comparison of kinetic parameters 11 K m (mM)k cat (s -1 )k cat /K m (s -1 mM -1 ) L-Cth LdCBL9 ( 2)1.2 ( 0.1)0.13 ( 0.03) EcCBL ( 0.01)34.1 ( 0.6)190 (10) SaCSL L-Cys LdCBL--- TdCSL SaCSL ( 0.02)109 ( 2)220 ( 10) L-Cys 2 LdCBL EcMCBL ( 0.1)-- Table 1: Comparison of kinetic parameters for L-Cth, L-Cys 2 and L-Cys substrates. Kinetic parameters determined here are compared to values for E. coli cystathionine - lyase (EcCBL), E. coli MalY Regulon/cystathionine -lyase (EcMCBL), Streptococcus anginosus C-S lyase (SaCSL) and Treponema denticola C-S lyase (TdCSL). Errors are provided where available. Although EcMCBL has been reported as catalyzing the - elimination of L-Cth, no values were available for the kinetic parameters of this reaction. 12 PDB IDFunctionOrganism% identity Z score RMSDK m (mM) K cat (s -1 ) 3NRAPutative AspAT Rhodobacter sphaeroides GDEAspATPyrococcus horikoshii J32AspATPhormidium lapideum DJUAroATPyrococcus horikoshii BJWAspATThermus thermophilus ASLAspATEscherichia coli Table 1. Bioinformatics Comparison of Protein Homologues of 3NRA. Percent identity was derived from PSI-BLAST alignments of primary structure and Z scores and RMSD values were calculated via the DALI server. Values listed are relative to 3NRA. The kinetic parameters with aspartate as substrate are included for those proteins that have been assayed for AspAT activity. 13 Troubleshooting and negative data Figures you may want to include 14 Figure 4. Rate dependence on concentration of RsAspAT. Activity is shown in Tris buffer and phosphate buffer. Substantially greater rates were observed in the Tris buffer than in phosphate buffer, in agreement with other investigators 5 ; however, rate dependence on enzyme concentration is observed in phosphate buffer as well. 15 16 Figure 7. Progress Curve of Enzyme Activity. Using an alanine dehydrogenase coupled assay, the POI was tested for aspartate decarboxylase activity 20 o C. The positive control was an aspartate decarboxylase from Bacillus cereus at 2.5 U/mL. The data suggests that the protein is not an aspartate decarboxylase. 17 Kinetics Data 18 Figure 11. Michaelis-Menton kinetics observed with aspartate. An MDH- coupled assay was performed to determine aspartate activity. Michaelis-Menton kinetics were observed. The data were plotted and fit with OriginPro to a hyperbolic function to determine the binding affinity (K m ) and maximum reaction velocity (V max ) for RsAspAT with aspartate.. Figure 12. Lineweaver-Burk analysis of RsAspAT activity with aspartate. The K m and V max were determined from the line equation and are reported above. Data were plotted and fit using OriginPro software. Should have error bars on LB Since the MM didnt have a lot of points and never reached V max, the LB or HW are a good way to substantiate the fit/kinetic parameters. Fonts could be larger 19 Figure 6. Michaelis-Menten kinetics of aspartate with POI. The activity of POI at both room temperature (21C; ), and at 37C (). This data shows that the overall rate for aspartate catalysis occurs faster at room temperature than at 37C. At room temperature, the k cat is 7.2 mM and the V max is 0.72 s -1. At 37C, the k cat is 1.5 s -1 and the V max is 0.15 M /s. 20 Figure 7. Kinetics of POI with - ketoglutarate and POI at room temperature (21C). The concentration of POI was constant as -ketoglutarate was varied from 0 to 5 mM. Kinetic data at -ketoglutarate concentrations greater than 1mM did not follow Michaelis-Menten fit which suggests possible substrate inhibition with higher concentrations of -ketoglutarate. 21 22 Other Data pH studies, substrate screens, inhibitors, etc. 23 Figure 1. A) Photo of Calvin Mura, born to Professors Columbus and Mura, and B) his basic biometrics. 24 Figure 9. Michaelis-Menten kinetics in the presence of aminooxyacetate inhibitor. Aspartate was increased in a gradient in the absence () and presence () of 5 mM aminooxyacetate at 21 C. Aminooxyacetate shows significant mixed inhibition of POI activity with aspartate as a substrate. Aminooxyacetate 25 Figure 5. Overlaid Michaelis-Menten plots for the L-alanine (A) and -ketoisovalerate (B) inhibition data. The uninhibited data is and the inhibited data are shown for each plot. Data points the average of three trials and parameters were extrapolated via nonlinear regression with the Michaelis-Menten equation in OriginPro. The inhibition by L- leucine was competitive with respect to L-alanine and uncompetitive with respect to - ketoisovalerate. Might be nice to see LB plot Should report new K m and V max Can calculate K i and compare to literature values 26 Fig. 7 Lineweaver Burk graph of enzyme activity with and without glycine. Both sets of data were fit via linear regression yielding line equations of y = x (R 2 = 0.985) with glycine and y = x (R 2 = 0.993) without glycine. The K m and V max, determined from each line equation, show that glycine changes the K m but not the V max. Glycine is therefore acting as a competitive inhibitor with a K I calculated to be 0.61 mM. n = 3. 27 Fig. 8 BxDSD kinetic parameters determined at varying pH. The data was fit with the hyperbolic Michaelis-Menten equation. BxDSD shows better activity at lower temperature and neutral pH. n = 3. 28