Progress 10/01/09 to 09/30/14

Outputs
Target Audience: Animal health investigators involved in infectious diseases. KAES life scientists and educators working in an area of structure activity relationship studies using NMR instrumentation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training and professional development for the following members: Dr. Daisuke Takahashi, Post-Doctoral Associate Dr. Hongzhou Huang, Graduate Student Dr. Sanjeev Shukla, Post-Doctoral Associate Ms. Yanji Bai, Graduate student Mr. Alvaro Herrera, Graduate Student How have the results been disseminated to communities of interest? Research publications, oral and poster presentations at national or international meetings What do you plan to do during the next reporting period to accomplish the goals? We will continue to assist AES researchers with NMR studies on hydrogel forming peptides, modified starch, serine and cysteine proteases, insect cuticles proteins, antimicrobial and channel forming peptides, and other on-going projects. We will continue our grants funding efforts to add a high-field NMR system (600 or 700 MHz) in our facility. For this purpose we will prepare grant proposals for submission to either NSF or NIH. We are at a serious disadvantage if we are not current in the methodologies being developed and made available to the agriculture NMR community particularly in our neighboring universities (OSU, ISU and UNL have these methodologies available to their faculty).

Impacts
What was accomplished under these goals? NMR studies on hydrogel forming peptide:- In a collaborative study we have used nuclear magnetic resonance (NMR) spectroscopy to monitor the conformational properties of a hydrogel-forming peptide FLIVIGSIIGPGGDGPGGD (h9e) during hydrogelation in different dimethylsulfoxide (DMSO)/H2O solutions. The peptide shows nanofiber morphologies in DMSO/H2O solution with a ratio lower than 4:1. Increased water percentage in the solution enhanced the hydrogelation rate and gel strength. 1D and 2D proton nuclear magnetic resonance (NMR) and electron microscopy studies performed on the peptide in DMSO/H2O solution with different ratios indicate that the peptide monomer tends to adopt a more helical structure during the hydrogelation as DMSO/H2O ratio is reduced. Interestingly, at the same DMSO/H2O ratio, adding calcium ions not only promotes peptide hydrogelation and gel strength, but also leads to special shear-thinning and recovery properties of the hydrogel. NMR Structural and functional studies on norovirus proteases:- With a long-term goal toward the development of protease inhibitors with broad-spectrum activity, we have characterized structural and dynamics properties of a noro virus protease (NVPro, 181 AA). Our NMR studies suggest that the overall fold of NVPro solution structures agrees well with that of the crystal structures, consisting of incomplete beta-barrel N-domain and anti-parallel beta-barrel C-domain with active site residues (H30, E54, C139) being located within a cleft between two domains. A notable difference is an additional very short beta-strand structure (V72-E74) observed in liker region connecting N- and C-domains. Study of Octenyl Succinic Anhydride(OSA) modified waxy maize starch by NMR:- OSA modified starch are more resistant to enzyme digestion and therefore this modification increases the level of slowly digestible and resistant starch. The OSA reagent contains a double bond and configuration of OSA in modified starch influences the properties of the end product. In this collaborative project, Dr. Shi's group is using high resolution one and two dimensional homo and heteronuclear NMR spectroscopy to determine the detailed structural characteristics of OSA reagent and OSA-modified waxy maize starches having different level of substitutions. The information gained in this study is needed to relate the structures of modified starch and their functional properties such as enzyme digestibility. Structure-function studies on peptide nanomaterials by NMR:- Two 15-residue peptide sequences, KhK (KKKFLIVIGSIIKKK) and Alternating Kh (KFLKKIVKIGKKSII), were designed for the purpose of determining the role of peptide sequence on solution morphology and conformation. Despite the same amino acid content, KhK solutions exhibited alpha-helical content at acidic pH, while Alternating Kh solutions demonstrated primarily random coil character as determined by circular dichroism and 2D- 1H-1H nuclear magnetic resonance spectroscopy. Further analysis by TEM showed marked differences in the peptide solution morphology. Peptide particle aggregation and fiber formation were significantly affected by solvent composition and pH values for both peptide sequences. Circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy demonstrated that random coil character increased at basic pH for KhK. In Alternating Kh circular dichroism spectra, random coil conformations were predominant at acidic pH. At basic pH, Alternating Kh exhibited an increase in beta-sheet character. The goal of these structural studies is to determine the contribution from amino-acid sequence and relationship between secondary structure and peptide morphology. Such information is valuable for tailoring protein nanomaterials for use in adhesive, biomedical, and pharmaceutical applications.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Guragain, Y., Herrera, A., Vadlani, P., and Prakash, O. (2014) Lignins of Bioenergy Crops. Natural Products Communications. 9: in press.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Animal health investigators involved in infectious diseases. KAES life scientists and educators working in are of structure activity relationship studies using NMR instrumentation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training and professional development for the following members: Dr. Daisuke Takahashi, post-doctoral associate Dr. Yunjeong Kim, research faculty How have the results been disseminated to communities of interest? 1. Research publications and poster presentations at national or international meetings 2. Oral presentations at regional and international conferences and research meetings. What do you plan to do during the next reporting period to accomplish the goals? We plan to carry out further studies to understand the mechanism of function of norovirus protease. We will continue to assist AES researchers with NMR studies on hydrogel forming peptides, modified starch, serine and cystein proteases, insect cuticles proteins, antimicrobial and channel forming peptides, and other on-going projects. We will continue our grants funding efforts to add a high-field NMR system (600 or 700 MHz) in our facility. For this purpose we will prepared grant proposals for submission to either NSF or NIH. We are at a serious disadvantage if we are not current in the methodologies being developed and made available to the agriculture NMR community particularly in our neighboring universities (OSU, ISU and UNL have these methodologies available to their faculty).

Impacts
What was accomplished under these goals? Norovirus protease is an essential enzyme for a proteolytic maturation of norovirus nonstructural proteins. The protease, therefore, has been implicated as a potential target for antiviral drug development. Though high resolution X-ray structures have given a wealth of information about how this protease interacts with its substrate, it is unclear how the fluidity of certain residues in binding pocket plays a role in substrate recognition as well as overall catalytic activity of norovirus protease. To understand the dynamics of the substrate binding pocket first we determined the solution NMR structure and studied the backbone dynamics of the protease from Norwalk virus (NVpro), a prototype strain of norovirus. The C-terminal domain, which is responsible for substrate binding, shows significant structural variation in our solution NMR structure when compared to crystal structures. Also 15N spin relaxation and CPMG based relaxation dispersion analyses show wide range of time scale motion for residues in the C terminal domain. In particular, the long loop spanning residues T123-G133 shows fast motion (ps-ns), and the residues in the bII-cII region forming the large hydrophobic pocket (S2 site) undergo us-ms time scale motion indicative of conformational exchange on slower time scales. Our studies suggest an important role these residues may play in substrate recognition.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Takahashi, D., Hiromasa, Y.; Kim, Y., Anbanandam, A., Yao, X; A. Chang, K. and Prakash, O. (2013). Structural and dynamics characterization of norovirus protease. Protein Science. 22: 347-357.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Peptide-based hydrogels are attractive biological materials for biological applications in tissue engineering, and drug delivery. Study of their self-assembly pathways from their monomer structures is important not only for undertaking the rational design of peptide-based materials, but also for understanding their biological functions and the mechanism of many human diseases relative to protein aggregation. In a collaborative study we have used nuclear55 etic resonance (NMR) spectroscopy to monitor the conformation properties of a hydrogel-forming peptide during hydrogelation in different dimethylsulfoxide (DMSO)/H2O solutions. The peptide shows nanofiber morphologies in DMSO/H2O solution with a ratio lower than 4:1. Increased water percentage in the solution enhanced the hydrogelation rate and gel strength. 1D and 2D proton nuclear magnetic resonance (NMR) and electron microscopy studies performed on the peptide in DMSO/H2O solution with different ratios indicate that the peptide monomer tends to adopt a more helical structure during the hydrogelation as DMSO/H2O ratio is reduced. Interestingly, at the same DMSO/H2O ratio, adding calcium ions not only promotes peptide hydrogelation and gel strength, but also leads to special shear-thinning and recovery properties of the hydrogel. Without changing the peptide conformation, calcium ion binds to the charged Asp residues and induces the change of interfiber interactions that play an important role in hydrogel properties. PARTICIPANTS: Collaborators- Hongzhou Huang, Alvaro I. Herrera, and Xiuzhi S. Sun TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
These NMR structural studies may help us to understand the assembly pathways of peptide monomers into their corresponding nanofiber structures and may lead to design new biological materials with controlled stiffness and shearing-respondent behaviors.

Publications

• Huang, H., Herrera, A., Luo, Z., Sun, X., and Prakash, O. (2012) Structural transformation and physical properties of a hydrogel forming peptide studied by NMR, TEM and dynamic rheomter Biophysical Journal 103: 979-988.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Noroviruses are the leading cause of acute food- or water-borne gastroenteritis outbreaks in humans and are classified as NIAID (National Institute of Allergy and Infectious Diseases-NIH) category B priority pathogens. However, no vaccine or antiviral is currently available for norovirus infection. Noroviral RNA genome is composed of three open reading frames (ORFs), and the ORF1 encodes a polyprotein that is cleaved by the viral 3C-like cysteine protease (Pro) into 6 non-structural proteins, which makes the Pro as an essential component for the viral replication as well as an attractive target for antiviral drug development. We have initiated the NMR studies of structural determinants for substrate specificity of noroviral proteases (NVPro). With a long-term goal toward the development of protease inhibitors with broad-spectrum activity, we performed 1D, 2D and 3D-NMR experiments to solve the solution structure of Noroviral protease (181 AA). Our NMR studies suggest that the overall fold of NVPro solution structures agrees well with that of the crystal structures, consisting of incomplete beta-barrel N-domain and anti-parallel beta-barrel C-domain with active site residues (H30, E54, C139) being located within a cleft between two domains. A notable difference is an additional very short beta-strand structure (V72-E74) observed in liker region connecting N- and C-domains. PARTICIPANTS: Collaborators- Yunjeong Kim, Asokan Anbanandam and Kyeong-Ok Chang TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We hope that the solution NMR structure studies will help us in designing the potential inhibitors of the protease.

Publications

• Takahashi, D., Kim, Y., Chang, K., Anbanandam, A. and Prakash, O. (2011).Backbone and side-chain 1H, 15N, and 13C resonance assignments of Norwalk virus protease. Biomolecular NMR Assign (in press).

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Two 15-residue peptide sequences, KhK (KKKFLIVIGSIIKKK) and Alternating Kh (KFLKKIVKIGKKSII), were designed for the purpose of determining the role of peptide sequence on solution morphology and conformation. Despite the same amino acid content, KhK solutions exhibited alpha-helical content at acidic pH, while Alternating Kh solutions demonstrated primarily random coil character as determined by circular dichroism and 2D- nuclear magnetic resonance spectroscopy. Further analysis by TEM showed marked differences in the peptide solution morphology. Peptide particle aggregation and fiber formation were significantly affected by solvent composition and pH values for both peptide sequences. Circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy demonstrated that random coil character increased at basic pH for KhK. In Alternating Kh circular dichroism spectra, random coil conformations were predominant at acidic pH. At basic pH, Alternating Kh exhibited an increase in beta-sheet character. PARTICIPANTS: Individuals: Om Prakash (PI), Daisuke Takahashi (Postdoctoral), Alvaro Herrera (Graduate Student), Huaien Dai (Graduate Student), Yong-Xi Gong (Research Assistant), Jason Lauer (Graduate Student), Debarishi Banerjee (Graduate Student), Denton Shanks (Undergraduate Student), Jeanne Shera (Postdoctoral), and Yanji Bai (Graduate student). Partner Organization: NIH. Collaborators- Xiuzhi Sun (Grain Sciences and Industry), Dolores Takemoto (Biochemistry), and Yong-Cheng Shi (Grain Sciences and Industry). Training: training in structural biology research for one undergraduate, five graduate and two postdoctoral students. TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The ability of peptides to spontaneously form ordered structures at the nanoscale will make them promising building blocks for nanomaterials.

Publications

• Shera, J. N., Takahashi, D., Herrera, A., Prakash, O., and Sun, X. (2010). Morphology and Structural Properties of pH-Responsive Amphiphilic Peptides. Journal of Nanoscience and Nanotechnology, 10:7981-7987.
• Lauer, J., Banerjee, D., Shanks, D., Dai, H., Gong, Y. X., Prakash, O., Takemoto, D. J. (2010). NMR/Function Relationship of Peptide Corresponding to the C1B1 region of PKCγ Protein & Peptide Letters, 17:1-10
• Bai, Y., Shi, Y., Herrera, A. I., and Prakash, O. (2010). Study of octenyl succinic anhydride modified waxy maize starch by nuclear magnetic resonance spectroscopy. Carbohyrate Polymers (in press).

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Four chemically unique phosphate ester forms were detected by P31-NMR when wheat starch was reacted with sodium trimetaphosphate (STMP), sodium tripolyphosphate (STTP) in sodium hydroxide starting at pH 11.5. Less than half of the total phosphate ester was in the form of cross-linked starch diester. These phosphate esters of starch were stable at pH 9.0 and became somewhat unstable at pH 10 and 11. At pH 12, the level of substituted phosphate esters decreased ~22% but distarch monophosphate esters (cross-linked bonds) increased ~12%. Even though the total amount of phosphate esters significantly degreased after the starch was treated at pH 12.00, the total dietary fiber (TDF) of the starch barely changed, suggesting that the level of the cross-linked phosphate diesters play a key role in making the phosphorylated starch resistant to alpha-amylase and amyloglucosidase digestion PARTICIPANTS: Collaborators- Yong-Cheng Shi and Paul A. Seib TARGET AUDIENCES: Life scientists and educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We hope that the chemical modifications of starch, such as phosphorylation, may be used to change its functionality and digestibility.

Publications

• Sang, Y, Seib, P., Herrera, A., Prakash, O., and Shi, Y. (2009) Effects of Alkaline Treatment on Structure of Phosphorylated Wheat Starch and its Digestibility. Food Chemistry. (in press).