Progress 04/15/15 to 04/14/17
Outputs
Target Audience:The target audience are entomologists, biochemists, plant scientists, agricultural scientists, and structural biologists. In these disciplines, students, scientists and public are part of the target audience. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for young scientists at undergraduate, graduate and postdctorate levels providing opportunities to learn molecular biology (cloning, mutagenesis), protein chemistry/biochemistry (overexpression of protein in bacteria, purification using varipus techniques such as dialysis, ion-exchange chromatography, and size sxclusion cheromatohraphy using state-of-the-art instruments such as AKTA FPLC chromatography system, biophysical chemistry including fluorescence and high-resolution solution-state NMR and computational chemistry (NMR data processing, assignments etc.). Not only these young scientists were trained but they also presented posters at various scientific conferences. How have the results been disseminated to communities of interest?The results have been presented at various scientific forums including conferences such as ACS (American Chemical Society), Biophysical conferences and presentation at various land-grant institutions and scientists working in entomology and agriculture/pest related areas. A mansucript has already been prepared that will be submitted to peer reviewed scientific journal for publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The goal of this project is to understand the mechanism of ligand binding and release at the molecular level. The following specific questions on ligand binding and release mechanism were addressed: (i) How does the C-terminus that is unstructured at high pH switch to a very structured alpha-helix at low pH and occupy the hydrophobic binding pocket? (ii) Which factors determine and control the transition of this switch? (iii) Does one or more of the three pH-sensitive residues present in the C-terminus play a role in this pH-driven reversible coil-helix transition for ligand release in ApolPBP1 at low pH? To test our hypothesis that these three charged residues (D132, E137 and E141) of ApolPBP1 either alone or in combination play a role in the reversible pH-driven coil-helix transition, a systematic mutagenesis study was carried out, whereby each charged residue was replaced by its neutral counterpart, either alone or in combination. The effects of the mutation/s on the protein conformation and ligand binding were characterized by nuclear magnetic resonance (NMR) and fluorescence studies respectively. Results (i) Themutant PBPs were overexpressed in bacteria and purified to homogeneity using dialysis, anion exchange and size exclusion chromatography. The mutant proteins generated were: three single mutants (D132N, E1347Q, E141Q), two double (D132N/E137Q and E137Q/E141Q), and one triple (D132N/E137Q/E141Q) mutant (3). To correlate the effect of mutation/s on protein conformation, biophysical studies were carried out using high-resolution solution NMR. Effect of Point Mutation on ApolPBP1 Conformation at High pH The effect of point mutation of D132N or E137Q or E141Q had no effect on ApolPBP1 conformation at high pH of 6.5. The 2D HSQC spectra of each of these single mutant proteins closely resembled that of wild type protein. Each single mutant protein displayed "open" or "B" conformation at high pH of 6.5. In the "open" conformation, the C-terminus is outside the hydrophobic cavity as a coil exposed to the solvent leaving the binding pocket open for ligand binding. Effect of Point Mutation on ApolPBP1 Conformation at Acidic pH Although point mutation of Asp132 or Glu137 or Glu141 does not affect the overall conformation of ApolPBP1 at high pH, their effect at acidic pH of 4.5 is very interesting. Unlike the ApolPBP1wt, which switches to closed/A conformation at low pH, each single mutant protein exhibited a mixture of both open/B conformation (similar to C-terminus truncated ApolPBP)and closed/A conformation (similar to ApolPBP1wt). Effect of Double Mutations on ApolPBP1 Conformation at High pH To examine the synergistic effect of two charged residues when mutated simultaneously on protein conformation and function, we produced two double mutants where either Asp132 and Glu137 (D132N/E137Q) or Glu137 and Glu141 (E137Q/E141Q) were mutated. The HSQC spectra of both the double mutants, ApolPBP1D132N/E137Q and ApolPBP1E137Q/E141Q exhibited the characteristic open conformation pattern of the wild type protein at pH 6.5 as shown for ApolPBP1E137Q/E141Q. Effect of Double Mutations on ApolPBP1 Conformation at Acidic pH 2D HSQC spectra of mutation of first two acidic residues i.e. Asp132 and Glu137 at pH 4.5 matched well with those of the single mutant proteins displaying both open and closed conformations. Mutation of these two residues together did not exhibit any synergistic effect on protein conformation than what was already observed for three single mutants at low pH. However, very interestingly, the impact of mutation of later two acidic residues i.e. Glu137 and Glu141 on the conformation and consequently the function of the protein was dramatically different. 2D HSQC spectra of ApolPBP1D137Q/E141Q exhibited only open conformation at acidic pH. It was very clear from the NMR data that mutation of both Glu137 and Glu141 simultaneously did knockout the reversible pH-driven coil-helix transition of the C-terminus keeping the protein in open conformation even at low pH very similar to C-terminus deleted ApolPBP1. Thus, the mutated C-terminus is unable to occupy the hydrophobic pocket as the 7th helix to displace the ligand at low pH in contrast to the ApolPBP1wt. Effect of Triple Mutations on ApolPBP1 Conformation at High pH As revealed by 2D HSQC NMR data, the triple mutant protein behaved very similarly as all other mutant proteins at high pH exhibiting open/B conformation similar to that of the wild type protein. At acidic pH, the fingerprint region in the 2D HSQC of the triple mutant protein resembled that of the C-terminal deleted proteinand E137Q/E141Q double mutant protein exhibiting open/B conformation only without undergoing pH-induced reversible coil-helix transition. These results indicate that no further effect on the protein conformation with the mutation of Asp132 along with Glu137 and Glu141. Based on our NMR characterization of three single mutant proteins, it is clear that all the three charged residues play important roles in the stabilization of the newly formed C-terminal helix (α7) inside the hydrophobic pocket of ApolPBP1 at low pH. However, Glu137 and Glu141 play a very critical role since mutation of these two residues is enough to disrupt helix-7 binding interactions inside the hydrophobic pocket. As a result the C-terminus stays outside the pocket in the extended conformation. Effect of Ligand on the Conformation of ApolPBP1 D132N/E137Q/E141Q For ligand titration studies, ApolPBP1triple mutant protein was delipidated to remove the endogenous ligand of the bacterial expression system by incubating the pure protein with lipidex resin at pH 4.5. Very interestingly, the {1H, 15N} HSQC spectrum of delipidated triple mutant protein matched very well with that of the undelipidated triple mutant suggesting that the protein remains in bound conformation even when the hydrophobic binding pocket is empty. NMR titration studies were carried out at pH 6.5 using palmitic acid (a fatty acid that binds to ApolPBP1). Protein:palmitic acid ratios were varied from 1:0 to 1:10. Ligand titration studies revealed that the triple mutant protein did not undergo any conformational change upon ligand binding. Very interestingly, unlike the two sets of peaks observed for ApolPBPwt protein during ligand binding, the mutant protein had only one set of resonances . The affected peaks chemical shift positions shifted gradually with the addition of increasing amounts of ligand. Additionally, the mutant protein did not reach saturation even with a 10-fold molar excess of ligand when the ApolPBPwt could achieve saturation at 3-fold excess of ligand (2). Taken together, these observations suggest that the mutant protein binds ligand with significantly lower affinity and has a shorter lifetime in the bound-state giving rise to only one set of NMR resonances. The average dissociation constant (Kd), calculated based on the titration curve for several peaks, is 1.20 ± 0.326 mM while the reported dissociation constant (Kd) for ApolPBPwt is 50 nM. AMA Binding Assay with Fluorescence The affinity of AMA (1-aminoanthracene) for ApolPBP1 and the six different mutant proteins (three single, two double and one triple mutant) were compared under identical experimental conditionsat pH 6.5 and 4.5. At pH 6.5, all the mutants of ApolPBP1 lost nearly 65% affinity for AMA compared to that of the wild-type protein. At pH 4.5, the fluorescence intensity is further reduced for all of the mutants except for one double mutant (ApolPBP1E137Q/E141Q) and the triple mutant (ApolPBP1D132N/E137Q/E141Q), which showed relatively higher binding compared to the ApolPBP1. The behavior of the double (ApolPBP1E137QE141Q) and triple (ApolPBP1D132NE137QE141Q) mutants in the AMA assay is consistent with what was observed in binding studies with NMR.
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2017
Citation:
Mohanty, S., Mazumder, S., Chaudhary, B., Dahal, S. and Essandoh, M. (2017) Probing the Mechanism of pH-Driven Reversible Coil-Helix Transition in the C-terminus of Antheraea polyphemus Pheromone-Binding Protein1, (Under preparation, to be submitted to EMBO Journal).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Suman Mazumder, Smita Mohanty, Pheromone Binding Protein: Structural & Mechanistic Insight into insect olfaction, 4th Annual Symposium on Structural Biology at University of Oklahoma - June 14, 2016.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Suman Mazumder, Bharat Chaudhary, Salik Dahal, Smita Mohanty, ROLE OF ACIDIC RESIDUES IN C-TERMINUS TAIL OF ANTHERAEA POLYPHEMUS PBP1 IN LIGAND BINDING AND RELEASING, 62nd Annual Oklahoma Pentasectional Meeting of the American Chemical Society, March 24-25, 2017 Cameron University Lawton, OK
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
American Chemical Society Speaker for ACS local section in Midwestern University, Texas, November 9, 2015.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Invited Speaker at 2015 Great Plains Regional Annual Symposium on Protein and Biomolecular NMR,The University of Kansas, November 13-14, 2015
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
Speaker at the First Biophysics Symposium on Protein Structure, Dynamics and Function at OSU on May 21, 2015
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Invited Speaker at Stephenson Cancer Research Center, University of Oklahoma Health Science Center, June 3, 2016.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Smita Mohanty, Pheromone Perception: Structure & Function of Pheromone-Binding Protein, Invited speaker in Department of Biochemistry Department Seminar Series at Kansas State University, September 22, 2016
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Invited Speaker, Pheromone Perception in Moth, National High Magnetic Field Laboratory, Tallahassee (FL), October 17, 2016
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Smita Mohanty, Pheromone Perception: Structure and Function of Pheromone-Binding Protein, University of California, Merced, CA, March 24, 2017
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Progress 04/15/15 to 04/14/16
Outputs
Target Audience:The target audience are primarily entomologists, biochemists and plant scientists. Students, scientists and general public as well. Changes/Problems:The approach has not been changedbut there have been problems with NMR instrument, which is very crucial this project.The major problem that the PI has faced since 2015 (after the funding was made available) that the Varian 600 MHz NMR instrument in the Oklahoma State-wideShared (OSS) NMR Facility located in theDepartment of Chemistry at Oklahoma State University (OSU) broke down several times in 2014 and 2015 for months at a time. The manufacturer of this instrument Varian/Agilent shut down the manufacturing and support for these instruments in October 2014. Thus, fixing of any hardware or software problems has become very difficult, time consuming and expensive.This is theonly high-resolution solution NMR spectrometer in the state of Oklahomafor protein NMR. The Varian 600 MHz instrument at OSU is about 21 years old. Since there is no cryoprobe, the sensitivity is very low.The PI has spent considerable amount of time writing a Major Research Instrumentation (MRI) grant proposal to NSF for the acquisition of a Bruker 800 MHz NMR instrument fitted with cryoprobe. This proposal was submitted in January 12, 2016 and is currently under review with NSF. Problems with NMR data collection have become the major bottleneck with structural and conformational characterization of ApolPBP1 mutants for this project. The PI has tried to ship samples to another NMR facility in the country but waiting period to get time has been long. Also setting up of NMR experiment remotely and depending on others to place the sample in the magnet are the difficulties that we have faced. The second problem is that the postdoctoral fellow, responsible for NMR work left in December of 2015. What opportunities for training and professional development has the project provided?Two postdoctoral fellows (one is continuing, but the second postdoctoral fellow left in December of 2015) were trained on this project. Two graduate students and one undergraduate student (although not supported by the funding) but got training on recombinant protein expression and purification with this project. How have the results been disseminated to communities of interest?The PI has given invited lecture at various forums including invited seminars at various Universities,4 -year undergraduate colleages and at conferences. Postdoctoral fellows also have presented posters at various research symposium/workshop. The data will be published in peer reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?We plan to further charaterize the six different mutant proteins using complementary biophysical techniquessuch as Circular Dichroisim (CD) /FT-IR and possibly through molecular dynamics studies of at least onedouble and the triple mutants to understand their behavior better. We will also work on ligandrecognition aspect of the project.
Impacts
What was accomplished under these goals?
Progress has been made to Aim-1 of this project. To get a mechanistic insight into the pH-driven C-terminal switch, we have mutated the three charged residues in the C-terminal tetradeca peptide segment of the PBP producing single, double and triple mutant proteins. The three chargedresidues present in this peptide segment are: Asp132, Glu137 and Glu 141. To understandthe role if any of eachof thecharged residuein the pH-driven conformation switch of ApolPBP1, we mutated eachresidueto itsneutral but polar counter part (Asp to Asnand Glufor Gln). We produced 3 single mutants, two double mutants and one triplemutant proteins to correlate the effect of mutation of each residue, and the synergistic effect of mutation of more than one residue on the pH-driven conformational switch. Mutagenesis studies were carried out and each mutant protein wassequenced to confirm the mutation. Six different mutant proteins: three single mutant proteins, twodouble mutant proteins andone triple mutant proteinwere overexpressed in bacteria, purified to homogeneity using dialysis, anion exchange and size exclusion chromatography. Fluorescence binding assays werecarried out to compare the ligand binding affinities of these mutant proteins tothat of the wild-type protein under identical conditions at both high and low pH levels. Currently NMR experiments arecarried outto characterize the conformation of each mutant protein at both high pH (pheromone binding conformation in the presence of a ligand) and low pH (pheromone releasing conformation) levels although this part of the project ia takinglong due to problems faced with our NMR instrument as described below.
Publications
- Type:
Other
Status:
Other
Year Published:
2015
Citation:
Pheromone Binding Protein: Structural and Mechanistic Insight into Insect Olfaction
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