Progress 02/01/11 to 01/31/15
Outputs
Target Audience: Scientists, Academicians, and Entemologists. The published paper target scientists and academicians in general and Entomologists in particular. Changes/Problems: The goals of the project or the method or approach has not changed. The PI is relocating and the funding will be transferred. What opportunities for training and professional development has the project provided? The project provides opportunities for the students at all levels (high school, undergraduate, graduate levels) and scientists training opportunities in molecular biology, protein chemistry, biophysics and computational chemistry. The students are able to presnt the work in several conferences. How have the results been disseminated to communities of interest? The results have been presented by the PI, Dr. Smita Mohanty, as invited seminars in City University of New Yrok, Staten Island in February 2013, Oklahoma State University in May 2013 and in Wesleyan University in November 2013 . The graduate student, Suman Mazumder (who graduated in August 2013) also presented this work at two different conferences (American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting in 2011 and in 2013 and The Southeastern Regional Meeting of the American Chemical Society (SERMACS) in 2013. The results were also published ina peer reviewed journal of American Chemical Society, Biochemistry. The citation of published journal articles or abstracts are- Uma V. Katre, Suman Mazumder, and Smita Mohanty, Biochemistry, 52, 1037-1044 (2013). Moth Olfaction: A Model of Exquisite Sensitivity and Specificity, Smita Mohanty, Biochem Physiol 1:e106. doi:10.4172/bcpc.1000e106 (2012). (http://www.omicsgroup.org/journals/ArchiveBCP/previousissue). Suman Mazumder, Uma V. Katre and Smita Mohanty, Role of C-terminus tail in the ligand binding and release mechanism of Antheraea polyphemus pheromone-binding protein 1, American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting, 244, C165 (2013) Suman Mazumder, Uma V. Katre and Smita Mohanty, Role of C-terminus tail in the ligand binding and release mechanism of Antheraea polyphemus pheromone-binding protein 1, The Southeastern Regional Meeting of the American Chemical Society (SERMACS), Atlanta, Georgia, November 12-16, 2013 Suman Mazumder, Uma V. Katre and Smita Mohanty, Pheromone Binding Protein: Structural and Mechanistic Insight into Insect Olfaction, American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting, 304, B226 (2011) What do you plan to do during the next reporting period to accomplish the goals? This funding will be tranferred to another institution that the PI is moving. Hence the PI will not report through Auburn University any longer.
Impacts
What was accomplished under these goals?
Pheromone-binding proteins (PBPs) in lepidopteran moths selectively transport the hydrophobic pheromone molecules across the sensillar lymph to trigger the neuronal response. Moth PBPs are known to bind ligand at physiological pH and release it at acidic pH while undergoing a conformational change. Two molecular switches are considered to play a role in this mechanism: (i) Protonation of His70 and His95 situated at one end of binding pocket, and (ii) Switch of the unstructured Cterminus at the other end of the binding pocket to a helix that enters the pocket. We have reported previously the role of the histidine-driven switch in ligand release for Antheraea polyphemus PBP1 (ApolPBP1). Here we show that the C-terminus plays a role in ligand release and binding mechanism of ApolPBP1. The C-terminus truncated mutants of ApolPBP1 (ApolPBP1ΔP129-V142 and ApolPBP1H70A/H95AΔP129-V142) exist only in the bound conformation at all pH levels, and they fail to undergo pH- or ligand- dependent conformational switch. Although these proteins could bind ligands even at acidic pH unlike the wild-type ApolPBP1, they had ~4 fold reduced affinity towards the ligand at both acidic and physiological pH compared to that of ApolPBP1wt and ApolPBP1H70A/H95A. Thus, apart from helping in the ligand-release at acidic pH, the C-terminus in ApolPBP1 also plays an important role in ligand binding and/or locking the ligand in the binding pocket. Our results are in stark contrast to those reported for BmorPBP and AtraPBP, where C-terminus truncated proteins had similar or increased pheromone-binding affinity at any pH. This work was published in 2013 and the citation is- Uma V. Katre, Suman Mazumder, and Smita Mohanty, Biochemistry, 52, 1037-1044 (2013).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Uma V. Katre, Suman Mazumder, and Smita Mohanty,Structural insights into the ligand binding and releasing mechanism
of Antheraea polyphemus PBP1: role of the C-terminal tail,
Biochemistry, 52, 1037-1044 (2013).
Suman Mazumder, Dissertation for Ph.D., submitted to Auburn University in August 2013.
Moth Olfaction: A Model of Exquisite Sensitivity and Specificity, Smita Mohanty, Biochem Physiol 1:e106. doi:10.4172/bcpc.1000e106 (2012).
(http://www.omicsgroup.org/journals/ArchiveBCP/previousissue).
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Progress 02/01/12 to 01/31/13
Outputs
Target Audience: Scientists, Academicians, and Entemologists. The published paper target scientists and academicians in general and Entomologists in particular. Changes/Problems: The goals of the project or the method or approach has not changed. What opportunities for training and professional development has the project provided? The project provided opportunities for the students at all levels (high school, undergraduate, graduate levels) and scientists training opportunities in molecular biology, protein chemistry, biophysics and computational chemistry. The students were able to presnt the work in several conferences. How have the results been disseminated to communities of interest? The results have been presented by the PI, Dr. Smita Mohanty, as invited seminars in City University of New Yrok, Staten Island in February 2013, Oklahoma State University in May 2013 and in Wesleyan University in November 2013 . The graduate student, Suman Mazumder (who graduated in August 2013) also presented this work at two different conferences (American Society for Biochemistry and Molecular Biology (ASBMB) Annual Meeting in 2013 and The Southeastern Regional Meeting of the American Chemical Society (SERMACS) in 2013. The results were also published ina peer reviewed journal of American Chemical Society, Biochemistry. The citation is- Uma V. Katre, Suman Mazumder, and Smita Mohanty, Biochemistry, 52, 1037-1044 (2013). What do you plan to do during the next reporting period to accomplish the goals? The mechanism of C-terminal switch of ApolPBP1 protein will be investigated in detail. We have recently shown that the C-terminal tail is very critical in the ligand release. The switc of the unstructured C-terminal tail (residues 129-142) at pH above 6.0 to an helix that enters/occupies the pheromone binding cavity at pH below 5.0 is very critical for the release of the ligand near the olfactory neuron. The C-terminal truncated ApolPBP1 is not able to realese the ligand at low pH. The next step is to understand the mechanism by which the C-terminus (residues 129-142) is switched from an unstructured structure to helix and enters the binding pocket. Which amino acid residue/s in the C-terminal tail of ApolPBP1is/are responsible for this switch at low pH? What is the impact of mutation of any of these residue/s on the C-terminal switch? Will the ligand release or binding be affected upon such mutation? These are the questions that will be addressed in the next reporting period.
Impacts
What was accomplished under these goals?
Pheromone-binding proteins (PBPs) in lepidopteran moths selectively transport the hydrophobic pheromone molecules across the sensillar lymph to trigger the neuronal response. Moth PBPs are known to bind ligand at physiological pH and release it at acidic pH while undergoing a conformational change. Two molecular switches are considered to play a role in this mechanism: (i) Protonation of His70 and His95 situated at one end of binding pocket, and (ii) Switch of the unstructured C-terminus at the other end of the binding pocket to a helix that enters the pocket. We have reported previously the role of the histidine-driven switch in ligand release for Antheraea polyphemus PBP1 (ApolPBP1). Here we show that the C-terminus plays a role in ligand release and binding mechanism of ApolPBP1. The C-terminus truncated mutants of ApolPBP1 (ApolPBP1ΔP129-V142 and ApolPBP1H70A/H95AΔP129-V142) exist only in the bound conformation at all pH levels, and they fail to undergo pH- or ligand- dependent conformational switch. Although these proteins could bind ligands even at acidic pH unlike the wild-type ApolPBP1, they had ~4 fold reduced affinity towards the ligand at both acidic and physiological pH compared to that of ApolPBP1wt and ApolPBP1H70A/H95A. Thus, apart from helping in the ligand-release at acidic pH, the C-terminus in ApolPBP1 also plays an important role in ligand binding and/or locking the ligand in the binding pocket. Our results are in stark contrast to those reported for BmorPBP and AtraPBP, where C-terminus truncated proteins had similar or increased pheromone-binding affinity at any pH. This work was published in 2013 and the citation is- Uma V. Katre, Suman Mazumder, and Smita Mohanty, Biochemistry, 52, 1037-1044 (2013).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Uma V. Katre, Suman Mazumder, and Smita Mohanty,Structural insights into the ligand binding and releasing mechanism of Antheraea polyphemus PBP1: role of the C-terminal tail, Biochemistry, 52, 1037-1044 (2013).
Suman Mazumder, Dissertation for Ph.D., submitted to Auburn University in August 2013.
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Progress 02/01/11 to 01/31/12
Outputs
OUTPUTS: Annual Progress Report of the AFRI Competitive Grant# 2011-65503-20030, Title: Pheromone Perception in Moth PI : Mohanty, S. This is the first annual progress report summarizing work performed during 2011. During this funding period, we have made progress for the aim #2 of the proposal which is to test the proposed model of ligand (pheromone) release by the Antheraea polyphemus pheromone-binding protein1 (ApolPBP1) at acidic pH near the olfactory neuron. It has been proposed by us and by other groups that the C-terminus switch along with the histidine switch is responsible for the ligand release near the olfactory neuron. We have recently mutated His70 and His95 to Ala and have shown that these histidines are indeed responsible for ligand release at low pH (1). Mutation of these two histidines (His70 and His95) to alanines prevented the release of the ligand at low pH. We proposed that His70 and His95 form a gate at one end of the ligand-binding cavity that opens at low pH due to charge repulsion between the positively charged side chains of these two histidines. Mutation to alanine keeps the gate permanently shut preventing the release of the ligand (1). However, the role of the C-terminus, which undergoes a switch from unstructured coil to a well formed helix and enters the binding cavity at low pH, in ligand release for ApolPBP1 is not known. To investigate the role of the ApolPBP1 C-terminus in ligand release, we produced 6 different mutants: the C-terminus-truncated mutants of ApolPBP1 (ApolPBP1ΔP129-V142) and ApolPBP1H70A/H95AΔP129-V142), ApolPBP1D132N, ApolPBP1E137Q, ApolPBP1E141Q, ApolPBP1D132N/E137Q/E141Q triple mutant. The residues P129-V142 form the C-terminus of ApolPBP1. The amino acid residues in the C-terminus that are labile to pH change are: Asp132, Glu137 and Glu141. Therefore along with the C-terminal truncated mutant, we investigated the role of these acidic residues in the formation of an helix at low pH. All the above mutants were produced through site-directed mutagenesis, overexpressed in E.coli, and purified to homogeneity for NMR and fluorescence studies. We collected 2D-{1H, 15N} HSQC experiments at both high and low pH for the ApolPBP1 (ApolPBP1ΔP129-V142) and ApolPBP1H70A/H95AΔP129-V142 along with investigation of ligand binding using fluorescence studies. 2D-{1H, 15N} HSQC NMR experiments on the other mutants are in progress. 1. Uma V. Katre, Suman Mazumdar, Rabi K. Prusti and Smita Mohanty, Ligand Binding Turns Moth Pheromone-Binding Protein into a pH Sensor: Effect on the Antheraea polyphemus PBP1 conformation, Journal of Biological Chemistry, 284 (46), 32167-32177 (2009). PARTICIPANTS: Suman Mazumder, graduate student, Mohiudeen Ovee, graduate student, and Smita Mohanty, principal investigator worked on this project. The project provides the above graduate students the opportunity to learn mutagenesis, protein chemistry (expression and purification), biophysics (NMR and fluorescence), and computational chemistry (NMR data processing and anlysis). They got the opportunity to learn and work on the state-of-the-art Bruker Avance 600 MHz NMR instrument fitted with a cryoprobe. The project certianly provides opoortunity to students at all levels (high school, undergraduate and graduate students) and postdoctoral fellows to learn modern biochemistry and biophysics. TARGET AUDIENCES: The results will help in the understanding of pheromone perception, binding and release mechanism in moth olfaction. This will help in the designing of pheromone mimetic for insect control. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the present work, we have investigated the role of C-terminus in the ligand binding and releasing mechanism of ApolPBP1. Truncation of C-terminus in ApolPBP1wt as well as ApolPBP1H70A/H95A resulted in proteins which exhibited the open or bound conformation regardless of pH and presence/absence of ligand. Although change of pH did not cause any drastic conformational change in these proteins from open (bound)to closed (free) conformation as was observed for undelipidated (ligand-bound) ApolPBP1wt, it did affect chemical shifts of several resonances indicating that local changes due to the effect of pH occurred in the protein. Delipidation also did not bring any major conformational change in these proteins in stark contrast to what was seen earlier for the ApolPBP1wt and ApolPBP1H70A/H95A at pH 6.5. Several resonances that had disappeared in the spectra of undelipidated proteins could be easily located in the spectra of delipidated proteins. These resonances again underwent line broadening and/or disappearance during ligand titration. Most of the resonances in all C-terminus truncated proteins exhibited intermediate-to-fast exchange phenomena on the NMR time scale during ligand titration, implying micromolar-to-millimolar affinities towards the ligand. Thus, while ApolPBP1wt and ApolPBP1H70A/H95A had nanomolar affinities towards ligands (characterized by the slow exchange seen for almost all resonances in HSQC during ligand titration), their C-terminus truncated counterparts had much lesser affinities. In fluorescence spectroscopic studies, ApolPBP1ΔP129-V142 and ApolPBP1H70A/H95AΔP129-V142 showed the ability to bind AMA even at low pH; unlike the wild type protein. However, their binding affinities at both pH 6.5 and 4.5 were greatly reduced by 2-6 folds as compared to ApolPBP1wt and ApolPBP1H70A/H95A. In conclusion, we have shown here that the C-terminus is essential for ligand release at low pH. Thus, even when the histidine gate is opened, the C-terminus truncated ApolPBP1 is unable to release the ligand and remain in bound or open conformation. Our data suggest that C-terminus switch from unstructured coil at high pH to helix at low pH that enters the binding cavity is necessary for the ejection of the ligand through the opened histidine gate.
Publications
- No publications reported this period
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