Biomolecular NMR Core Laboratory for Bioagricultural Sciences Research at KSU

BIOMOLECULAR NMR CORE LABORATORY FOR BIOAGRICULTURAL SCIENCES RESEARCH AT KSU

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

HATCH

Reporting Frequency

Annual

Accession No.

1004019

Grant No.

(N/A)

Cumulative Award Amt.

(N/A)

Proposal No.

(N/A)

Multistate No.

(N/A)

Project Start Date

Oct 1, 2014

Project End Date

Sep 30, 2019

Grant Year

(N/A)

Program Code

[(N/A)]- (N/A)

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
Biochemistry and Molecular Biophysics

Non Technical Summary
The biomolecular NMR core laboratory will assist KAES scientists in performing modern multidimensional NMR analyses to determine solution structure of proteins, carbohydrates, nucleic acids, peptides and other biomacromolecules in their functional states. This information will help researchers in designing biomaterials, pharmaceuticals, protein structures for plant and animal resistance factors to insects and diseases. The engineered proteins can be patented and will have the potential to promote a long term economic growth in Kansas. It is also noteworthy that this facility is one of the few NMR centers in the Midwest conducting structure and dynamics studies of peptides, proteins and other biomolecules. Prior to establishing this laboratory, KAES researchers were dependent on out-of-state facilities.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	2499	1040	40%
203	2499	1040	40%
712	2499	1060	20%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 202 - Plant Genetic Resources;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems);

Keywords

nuclear magnetic resonance (nmr) spectroscopy

Goals / Objectives
One of the main objectives of this biomolecular nuclear magnetic resonance core laboratory for bioagriculral sciences will be to assist KAES researchers in conducting nationally competitive research at Kansas State University in diverse areas of agriculture biochemistry, structure biology and in characterization of chemicals and their products in food, industrial, environmental and agriculture products samples.This NMR facility at K-State will initiate new NMR-linked grant proposals with KAES researchers, developing cutting-edge techniques and makeing sure that our students are educated and trained in using latest multinuclear, multi-dimensional NMR techniques.The Biomolecular NMR laboratory will provide and continue services of an expensive NMR instrumentation and solution structure determination infrastructure to KAES investigators which is difficult to acquire, operate and maintain by an individual investigator.This laboratory will also support KAES investigators in molecular modeling and dynamics of proteins to guide the design of future experiments, particularly those employing recombinant DNA technology to create new protein structures useful in developing resistance to insects and diseases in plants and animals, designing crops and animals with specific properties for special end uses and other useful agricultural commodities.

Project Methods
Brief introduction and literature review- The advent of faster computer systems with sophisticated microprocessors, highly sensitive cryogenic and micro-capillary NMR probes has transformed NMR spectroscopy into a method of choice for biochemists, molecular biologists, soil and food chemists, plant-pathologist, animal, grain and other agriculture scientists for detecting various chemicals and characterizing macromolecular structure in solution (Suzuki et al., 2001). The NMR measurements provide structural data as well as information on dynamics, conformational equilibria, folding and intra as well as intermolecular interactions (Kay, 2011; Palmer, 2004). The latest NMR methodology along with stable isotope labeling and transverse relaxation optimization technique (TROSY, Pervushin et. al, 1997) has made it feasible to determine the structure of proteins even larger than 100 kDa (Pellecchia, M. et al. 2002).At biomolecular NMR core laboratory, we use NMR methods for biomacromolecules, pioneered by Wuthrich, who won the 2002 Chemistry Nobel prize, and others (Wuthrich, K. 1986, 2003; Ikura et al., 1990; Bax et al., 1993; Kay, L. E. 2011) for 3D structure determination of biomacromolecules. The protocols for NMR structure determination can be summarized as follows: preparation of sample solution in a deuterated solvent or in water containing 10% D2O, the multi-dimensional NMR measurements, the assignments of NMR signals to individual atoms in the molecule, identification of secondary structure elements and conformational constraints (e.g. distances between hydrogen atoms), and calculation of the three-dimensional structure on the basis of the experimental constraints (Wuthrich, K. 1986; Bax et al. 1993; Kwan et al. 2011).Brief summary of previous work- This biomolecular NMR laboratory has expanded 1D and multidimansional NMR experiments described above into new protocols to solve the 3D solution structure of three new antimicrobial peptides from chicken (Xiao et al., 2008 and 2006; Bommineni et al. 2007); an antimicrobial peptide and a paralytic peptide from an insect Manduca sexta ( Dai et al., 2008 and Yu et al., 1999); a hydrogel forming peptide (Huang et al., 2012); a high potency pore-forming peptide (Herrera et al., 2010) and to characterize an antifungal metabolite produce by a new strain Pseudomonas aeruginosa PUPa3 ( Kumar et al. 2005). NMR spectroscopy has also been used to characterize Octenyl Succinic Anhydride(OSA) modified waxy maize starch (Bai et al., 2011) and phosphorylated cross-linked resistant starch (Sang et al.2007) and in identification of glycine betain in strawberry plants (Rajashekar et al., 1999). This laboratory has also applied multidimensional heteronuclear 2D and 3D NMR experiments such as heteronuclear multiple quantum coherence (HMQC; Bax et al., 1983), heteronuclear single quantum coherence - total correlation spectroscopy (HSQC-TOCSY, Oschkinat et al., 1994), NOESY - HSQC (Fairbrother et al., 1992) to solve the 3D solution structure of clip domains (124AA) from Manduca sexta prophenoloxidase activating proteinase-2, an enzyme involved in defense responses in arthropods (Huang et al. 2007) and a noroviral protease (181 AA), which play a critical role in viral replication (Takahashi et al. 2012 and 2013).NMR methods/experimental procedures to be utilized in carrying out above mentioned projects- For NMR measurements we will prepare 1-3 mM solution (0.3ml) of sample either in water containing 10% D2O or in a deuterated solvent. For NMR signal assignments this laboratory will use 2-D homonuclear spin correlated spectroscopy (COSY; BAX & Freeman, 1981) and 2-D total correlation spectroscopy (TOCSY; Bax & Davis, 1985a) followed by 2-D NMR experiments determining through space connectivities such as 2D proton - proton nuclear Overhauser effect spectroscopy (NOESY; Kumar, et al., 1980) and rotating frame Overhauser effect spectroscopy (ROESY; Bax & Davis, 1985b). Since the different secondary structure elements in a peptide, protein and macromolecules produce different NOESY signal patterns (Billeter, et al. 1982), therefore, NOESY spectra analyses will lead to the identification of secondary structure. The quantitative analyses of NOESY data will be used to estimate proton - proton distance constraints with the help of isolated spin pair approximation method. For the calculation of 3-D structure on the basis of the experimental constraints, this laboratory will use a computational approach developed by Clore et al., which combines distance geometry algorithms (Havel et al., 1983; Braun et al., 1985) dynamical simulated annealing (Nilges et al., 1988a and 1988b) and restrained molecular dynamics (Clore et al., 1989). The software modules: QUANTA/X-PLOR, CNS, MOLMOL and PyMOL will be used for structure calculation and molecular graphics (Bruenger, et al., 1998; Koradi et al., 1996; and DeLano 2005)We will perform relaxation measurements NMR experiments developed by Wagner et al. (Nirmala & Wagner, 1988, 1989) to determine the relaxation properties of alpha -carbon and amide Nitrogen atoms of backbone in a peptide or protein. We will determine order parameters for the macromolecules with the help of model-free formalism developed by Lipari, et al. (Lipari & Szabo, 1982a, 1982b) using rotational correlation time values. These studies will provide information about the flexibility and rigid region in a macromolecule on a time scale of nanosecond (Clore, et al. 1990). We will perform difference spectroscopy and heteronuclear single quantum coherence (HSQC; Bodenhausen & Ruben, 1980) measurements to study the protein interactions with other protein, peptide, and ligand. We will collect NMR data for freshly prepared protein or peptide sample in D2O and monitor the changes (NMR resonance frequency signal and intensity) occurring in amide proton as a function of time and pH. These experiments will be also useful for hydrogen exchange kinetics analysis and protein flexibility studies (Wagner, et al. 1982). For protein NMR studies, the recombinant forms of protein domains and/or proteins discussed above will be isotopically labeled with N15, and C13 as needed. We will perform 1D, 2D and 3D NMR experiments on these domains combined with a standard suite of triple resonance and residual dipolar couplings experiments (Ikura, 1990, and Lipsitz et al., 2004).The triple resonance experiments (yamazaki et al., 1994) include the CBCA(CO)NH, CA(CO)NH, H(CA)(CO)NH and HNCACB. For NMR structural studies on Phosphorylated wheat starch, we will perform 1D- 1H, 1D-broad band proton decoupled 13C and 31P, 2D- 1H-1H TOCSY, NOESY and 1H-13C HSQC NMR experiments in D2O to determine the structure of distarch monophosphate (DSMP), monostarch monophosphate (MSMP), monostarch diphosphate (MSDP), and cyclic monostarch monophosphate (cyclic-MSMP).Evaluation of action plan- Evaluation will be determined by observation of this facility usage in research projects described above, number of KAES researchers and students trained and development of the NMR-linked grant proposals submitted for extramural funding. The results of NMR studies carried out by KAES investigators at this center will be used for grant proposal preparation, national and international meetings presentation and for peer reviewed research articles publications. (Please see supplements I-IV for investigators assisted, research grant proposals funded with the support of biomolecular NMR facility, papers published and number of students educated/trained).

Progress 10/01/14 to 09/30/19

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. Hongzhou Huang, Graduate Student Dr. Sanjeev Shukla, Post-Doctoral Associate Ms. Yanji Bai, Graduate student Mr. Alvaro Herrera, Graduate Student Mr. Jordan Woehl, Graduate student Mr. Qasim Al Souhail, Graduate Stident Ms. Nicoleta Ploscari, Graduate Student Mr. Lynn Schrag, Graduate Student Mr. Nitin Mishra, Graduate Student Ms. Hawa Dembele, Under Graduate Student How have the results been disseminated to communities of interest?Research publications and presentations at national or international meetings 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 Biomolecular Nuclear Magnetic Resonance (NMR) Core Facility serves AES investigators and educators to conduct nationally competitive cutting edge research at Kansas State University in diverse areas of structural biochemistry, metabonomics, structure analyses of chemicals and their products in clinical, food, industrial, environmental and agriculture products samples. Our measurable objectives are:1. To perform modern multidimensional NMR experiments to identify and characterize chemicals in soil, plants, food and other agriculture products samples. 2. To study 3D solution structure and dynamics of carbohydrates, peptides, protein or protein domains that have been cloned and require knowledge of their 3D structure not only for understanding their function but intelligently implementing site-directed mutagenesis to create new protein structure useful in developing resistance to insects and diseases in plants and animals, designing crops with specific properties for special end uses and other useful agricultural commodities. 3. To develop NMR-linked grant proposals with AES scientists and make sure that our students and researchers are educated and trained in using latest NMR and 3D protein. 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 showed 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 indicated that the peptide monomer inclined 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 promoted peptide hydrogelation and gel strength, but also led 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 suggested that the overall fold of NVPro solution structures agreed 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 was 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 used 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 were useful 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 was to determine the contribution from amino-acid sequence and relationship between secondary structure and peptide morphology valuable for tailoring protein nanomaterials for use in adhesive, biomedical, and pharmaceutical applications. NMR Structural and functional studies on stress response peptides: In several moth species, proteolytic activation of plasmatocyte spreading peptide (PSP, also known as GBP for growth blocking peptide) stimulates hemocyte encapsulation. Homologs of GBP, including upstream ENF peptides (uENFs) and stress responsive peptides (SRPs) are identified in many insects including mosquitoes. Since most of these peptides are activated during stress conditions, such as wounding, infection, ligation, heat or cold shock, we adopted SRP as a general term to describe these structurally related molecules. To understand the multiple functions of Manduca SRP2 and its structure-function relationship, we initiated a two-dimensional proton-proton Nuclear Magnetic Resonance (NMR) spectroscopy studies to determine its solution structure. We have completed proton chemical shift assignments for this 29 amino-acids containing peptide using a set of 2D proton-proton through bond and through space correlation experiments. The tertiary structure of this peptide was determined and published. NMR Studies of a Novel Family of Serine Protease Inhibitors from S. aureus: Staphylococcus aureusis a ubiquitous and persistent pathogen of humans and livestock. The bacterium disrupts the host's innate immune system's ability to recognize and clear bacteria with optimal efficiency by expressing a wide variety of virulence proteins. Two single domain protein homologs (EapH1, EapH2) of the extracellular adherence protein (Eap) have been reported. Eap is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement and Neutrophil Serine Proteases (NSPs). EapH1 and EapH2 are also inhibitors of NSPs (Stapels et al. 2014), but lack the ability to inhibit the classical, and lectin pathways of the complement activation system. We acquired a series of 2D and 3D NMR spectra of EapH2 in solution. We completed 99% of expected non-proline backbone 1H, 15N, and 13C resonance assignments of EapH2 and determined secondary structure via the TALOS-N server. The assignment data were deposited in the BMRB data bank under accession number 27540. Recently we have been funded by an NIH award to purchase a high-field NMR system. We will be installing the new spectrometer and upgrading our current instrumentation during the next reporting period. We will continue to assist AES researchers with NMR studies on stress responsive peptides, a novel family of serine protease inhibitors from S. aureus, modified starch, insect cuticles proteins, antimicrobial and channel forming peptides, and other on-going projects.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: Schrag, L., Cao, X., Dembele, H., Liu, X., Al Souhail, Q., Kanost, M., Chen, J., Jiang, H., and Prakash, O. (2019) Expression and Characterization of Manduca sexta Stress Responsive Peptide-1; an Inducer of Anrtimicrobial Peptide Synthesis. Biochemistry and Molecular Biology 4(3): 42-52.
Type: Journal Articles Status: Published Year Published: 2019 Citation: Herrera, A., Dubey, A., Geisbrecht, B., Arthanari, H., and Prakash, O. (2019) Backbone resonance assignments of innate immune evasion protein EapH2 from the Staphylococcal aureus. Biomolecular NMR Assign 13:219-222.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dembele, H., Mating, M., Herrera, A., Park, Y., and Om Prakash (2019) NMR Solution Structure of Ecdysis Triggering Hormone from African Malaria Mosquito Anopheles gambiae. 2019-K-INBRE Symposium, Overland Park, KS, Jan. 19-20.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dembele, H., Mating, M., Herrera, A., Park, Y., and Om Prakash (2019) NMR Solution Structure of Ecdysis Triggering Hormone from African Malaria Mosquito Anopheles gambiae. American Chemical Society Student Affiliate Annual Research Forum, Manhattan, KS. May 4.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Schrag, L., Thevarajan, I., Liu, X., Zolkiewski, M., Prakash, O., and Chen, J. (2019) Structural and Functional Consequences of the Cancer-linked Mutations in the Intrinsically-Disordered Transactivation Domain of p53. ACS Midwest regional Meeting, Wichita, KS Oct. 16-19.

Progress 10/01/17 to 09/30/18

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: Mr. Lynn Schrag, Graduate Student Mr. Alvaro Herrera, NMR Research Assistant Ms. Nicoleta Ploscari, Graduate Student Ms. Hawa Dembele, Under Graduate Student How have the results been disseminated to communities of interest?Research publications and 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 stress responsive peptides, a novel family of serine protease inhibitors from S. aureus, modified starch, insect cuticles proteins, antimicrobial and channel forming peptides, and other on-going projects. Our grant proposal submitted to NIH for upgrading the current 500 MHz NMR instrumentation is under review. We will also 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 NSF-MRI or NIH shared instrumentation funding program. 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? PROGRAM: The Biomolecular Nuclear Magnetic Resonance (NMR) Core Facility serves AES investigators and educators to conduct nationally competitive cutting edge research at Kansas State University in diverse areas of structural biochemistry, metabonomics, structure analyses of chemicals and their products in clinical, food, industrial, environmental and agriculture products samples. Our measurable objectives are:1. To perform modern multidimensional NMR experiments to identify and characterize chemicals in soil, plants, food and other agriculture products samples. 2. To study 3D solution structure and dynamics of carbohydrates, peptides, protein or protein domains that have been cloned and require knowledge of their 3D structure not only for understanding their function but intelligently implementing site-directed mutagenesis to create new protein structure useful in developing resistance to insects and diseases in plants and animals, designing crops with specific properties for special end uses and other useful agricultural commodities. 3. To develop NMR-linked grant proposals with AES scientists and make sure that our students and researchers are educated and trained in using latest NMR and 3D protein structure determination methodology. IMPACT: The high-resolution nuclear magnetic resonance core facility for macromolecules provides valuable information to the KAES investigators to resolve many structural components and to quantify the changes produced in proteins or other molecules by chemical process. Such understanding is important for progress in medicine, nutrition, environmental concerns, and agricultural productions. NMR studies carried out at this laboratory provides new and detailed information about the molecular structure and dynamic behavior of proteins which in turn can offer important insight into processes such as protein gelation, protein modification, and protein folding etc. Molecular modeling and dynamic analyses based on NMR data guide AES researchers in designing future experiments, particularly those employing genetic engineering to create new proteins structure useful in developing resistance to insects and diseases in plants and animals, designing environment friendly materials such as biodegradable biopolymers, developing crops and animals with specific properties for special end uses and other useful agricultural commodities. The following paragraph summarizes the social and health impacts of our collaborative projects on structure-activity relationship studies of stress responsive peptides from insects and serine protease inhibitors from Staphylococcus aureus. Stress responsive peptides having the similarity with c-terminal sub-domain of human epidermal growth factor may play a critical role in oncogenesis. The structure-activity relationship studies may lead us to the development of novel inhibitors of epidermal growth factor receptor to be used in cancer treatment. The neutrophils are the primary type of white blood cell responsible for our body's innate response against S. aureus as well as a key driver of inflammatory processes in human diseases. Understanding how S. aureus inhibits neutrophil functions may reveal new approaches to preventing and/or treating staphylococcal infections. Our ongoing NMR structural studies on extracellular adherence proteins from S. aureus which act as potent inhibitors of neutrophil granule enzymes may aid us with a basic science foundation to future development of novel anti-inflammatory inhibitor therapeutics. Development of this core facility with the High-Field NMR Spectrometer as major equipment in the Department of Biochemistry has directly supported more than 49 grant applications for extramural funding. A number of NSF, NIH, Amer. Heart Assoc., Amer. Chem. Soc., Kansas Soybean Commission, KSU targeted excellence, and smaller awards have been funded. More than 55 researchers at KSU, KU, WSU, and other academic institutions including three industries have used this facility in their research program. Several of these KAES research projects involve investigation of proteins or protein domains that have been cloned and require knowledge of their three dimensional structure not only for understanding their function but intelligently implementing site directed mutagenesis to generate new potentially useful products. These engineered proteins can be patented and have the potential to promote a long term economic growth in the state. It is also noteworthy that this center is one of the few NMR laboratories in the Midwest conducting structure and dynamics studies of proteins and peptides. Manduca sexta stress response peptide-1, a 25-residue peptide with one disulfide bond (SRP1: FGVRVGTCPSGYVRRGTFCFPDDDY) functions as a cytokine to modulate immune responses. It is produced as an inactive precursor (proSRP1), which is converted to active SRP1 by proteolytic cleavage in response to invading pathogens. In addition to its role in immunity, SRP1 may control development or other physiological processes in this lepidopteran insect. Here we examined the expression profiles of SRP1 in terms of immune inducibility, tissue specificity, stress responsiveness, and development. We injected a chemically synthesized SRP1 into naïve larvae and detected up-regulation of some antimicrobial peptide genes. We solved the 3D structure of SRP1 in solution by two-dimensional 1H-1H NMR spectroscopy. SRP1 consists of two short β-strands at Y12−R15 and F18−F20, and one type-II β-turn at R15−F18 in its well-defined core and is stabilized by a covalent disulfide bond between C8 and C19. The SRP1 core adopts a fold similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF), suggesting that SRP1 may interact with EGF receptor-like molecules to trigger its biological function. These results have been submitted for publication in the Journal of Protein and Peptide Letters. Staphylococcus aureusis a widespread and persistent pathogen of humans and livestock. The bacterium expresses a wide variety of virulence proteins, many of which serve to disrupt the host's innate immune system from recognizing and clearing bacteria with optimal efficiency. The extracellular adherence protein (Eap) is a multidomain protein that participates in various protein-protein interactions that inhibit the innate immune response, including both the complement system and Neutrophil Serine Proteases (NSPs). The third domain of Eap, Eap3, is an ~11 kDa protein that was recently shown to bind complement component C4b and therefore play an essential role in inhibiting the classical and lectin pathways of complement. Since structural characterization of Eap3 is still incomplete, we acquired a series of 2D and 3D NMR spectra of Eap3 in solution. We have completed the backbone and side-chain resonance assignments of the Eap3. We have determined the secondary structure using TALOS-N prediction server. The assignment data is published in the Journal of Biomolecular NMR Assignments.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: G Schrag, L., Cao, X., al Souhail, Q., Kanost, M., Jiang, H., Prakash, O. (2018) NMR Solution Structure and Expression Profile of Stress Response Peptide-1: A Cytokine from Manduca sexta. 11Th Great Plains Regional Annual Symposium on Protein and Biomolecular NMR Oct. 12-13, Lawrence, KS.
Type: Journal Articles Status: Published Year Published: 2018 Citation: Herrera, A., Ploscariu, N., Geisbrecht, B., and Prakash, O. (2018) 1H, 15N, and 13C resonance assignments of the third domain from the Staphylococcal aureus innate immune evasion protein Eap. Biomolecular NMR Assign 1:175-178.. PMID: 29372458
Type: Journal Articles Status: Published Year Published: 2018 Citation: Jong, N., Ploscariu, N., Ramyar, K., Garcia, B., Herrera, A., Prakash, O., Katz, B., Leidal K., Nauseef, W., Kessel, K., Strijp, J., and Geisbrecht, B. (2018) A Structurally Dynamic N-terminal Region Drives Function of the Staphylococcal Peroxidase Inhibitor (SPIN), J. Bio.Chem. 293: 2260-2271.
Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Dembele, H., G Schrag, L., Liu, X., I Herrera, A. Chen, J., Prakash, O. (2018) Molecular Dynamics Studies of Stress Response Peptide-2 and its Mutants. 11Th Great Plains Regional Annual Symposium on Protein and Biomolecular NMR Oct. 12-13, Lawrence, KS
Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Thevarajan, I., G Schrag, L., Liu, X., Chen, J., Prakash, O., Zolkiewski, M. (2018) Structural and Functional Consequences of the Cancer-linked Mutations in the Transactivation Domain of p53. 11Th Great Plains Regional Annual Symposium on Protein and Biomolecular NMR Oct. 12-13, Lawrence, KS.
Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Dembele, H., Lynn G. Schrag, Xiaorong Liu, Alvaro I. Herrera, Jianhan Chen, and Om Prakash (2018) Molecular Dynamics Studies of Stress Response Peptide-2 and its Mutants. American Chemical Society Student Affiliate Forum, April 14, Manhattan, KS.
Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ploscariu, N., Herrera, A., Geisbrecht, B., and Prakash, O.(2018) NMR Structural and Binding Studies of SPIN (2018), ⿿NMR Structural Studies of the Extracellular Adherence Protein Domain- 4 at 28th International Conference on Magnetic Resonance in Biological System, Dublin, Ireland, August 21-26.

Progress 10/01/16 to 09/30/17

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: Mr. Lynn Schrag, Graduate Student Mr. Jordan Woehl, Graduate student Mr. Alvaro Herrera, NMR Research Assistant Ms. Nicoleta Ploscari, Graduate Student Ms. Hawa Dembele, Under Graduate Student How have the results been disseminated to communities of interest?1. Research publications and 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 stress responsive peptides, a novel family of serine protease inhibitors from S. aureus, modified starch, 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 NSF-MRI or NIH shared instrumentation funding program. 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? Manduca sexta stress response peptide-2 (SRP2) is predicted to be a 25-residue peptide (FGVK DGKCPSGRVRRLGICVPDDDY), which may function as an insect cytokine to regulate immune responses. Produced as an inactive precursor, endogenous proSRP2 is probably converted to active SRP2 by limited proteolysis in response to invading pathogens, along with prophenoloxidase and proSpätzle activation. Besides immunity, SRP2 may also control head morphogenesis or other developmental processes in this lepidopteran insect. In this work, we have examined the profiles of SRP2 gene expression in terms of immune inducibility, tissue specificity, and developmental changes. To gain insights into its functions, we chemically synthesized SRP2, injected the peptide solution into naïve larvae, and detected significant up-regulation of several antimicrobial peptide genes. We have determined the tertiary structure of SRP2 by two-dimensional 1H NMR spectroscopy. SRP2 has an ordered structure, which is composed of two short β-strands at Gly11 − Arg15 and Ile18 − Val20, one type-I' β-turn at Arg15 − Ile18, and a half turn at Cys8 − Ser10 in its well-defined core stabilized by a covalent disulfide bond between Cys8 and Cys19. These results are published in Protein and Peptide Letters. Further studies on possible relationships between the structure and function are under progress. The bacteriumStaphylococcus aureusis highly efficient in producing anti-inflammatory molecules that prevent the innate immune system from recognizing it as a pathogen and clearing it from the host. In the acute phase of inflammation, our immune system relies on neutrophils to clear the invading bacteria. Recently, novel classes of secreted proteins fromS. aureus, such asthe Extracellular Adherence Protein (EAP) and Staphylococcal Peroxidase Inhibitor (SPIN) proteins,have been identified as neutrophil inhibitors acting on Neutrophil Serine Proteases (NSPs) and myeloperoxidase (MPO) respectively. SPIN is a protein found only in Staphylococci, with no sequence homology to any known proteins. The solution NMR structural studies of SPIN provide a deeper understanding of its interaction with MPO. We have completed the backbone and side-chain resonance assignments of the SPIN and using their chemical shifts, we have determined the secondary structure using TALOS-N prediction server. The assignment data is published in the Journal of Biomolecular NMR Assignments. Further work on tertiary structure determination is under progress.

Publications

Type: Journal Articles Status: Published Year Published: 2017 Citation: Ploscariu, N., Herrera, A., Jayanthi, S. Kumar, T.K.S., Geisbrecht, B., and Prakash, O. (2017) Backbone and side-chain 1H, 15N, and 13C resonance assignments of a novel Staphylococcal inhibitor of Myeloperoxidase, Biomolecular NMR Assign. 11:285-288.
Type: Journal Articles Status: Published Year Published: 2017 Citation: Schrag, L.,Cao, X., Herrera, A., Wang, Y., Jiang, H., and Prakash, O. (2017) Solution structure and expression profile of an insect cytokine: Manduca sexta stress response peptide-2. Protein and Peptide Letters 24:3-11
Type: Book Chapters Status: Published Year Published: 2017 Citation: Schrag, L., Herrera, A., wang, Y., Jiang, H., and Prakash, O. (2017) Structure and function of stress responsive peptides in insects. Peptide-Based Drug Discovery: Challenges and New Therapeutics, Drug Discovery Series 59:438-451.

Progress 10/01/15 to 09/30/16

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: Mr. Lynn Schrag, Graduate Student Mr. Jordan Woehl, Graduate student Mr. Alvaro Herrera, NMR Research Assistant Mr. Qasim Al Souhail, Graduate Student Ms. Nicoleta Ploscari, 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 stress responsive peptides, novel family of serine protease inhibitors from S. aureus, modified starch, 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 NSF-MRI or NIH shared instrumentation funding program. 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? The pathogenic bacterium Staphylococcus aureus has evolved to actively evade many aspects of the human innate immune system by expressing a series of secreted inhibitory proteins. Among these, the extracellular adherence protein (Eap) has been shown to inhibit the classical and lectin pathways of the complement system. By binding to complement component C4b, Eap is able to inhibit formation of the CP/LP C3 pro-convertase. Secreted full-length, mature Eap consists of four ~98 residue domains, all of which adopt a similar beta-grasp fold, and are connected through a short linker region. Through multiple biochemical approaches, it has been determined that the third and fourth domains of Eap are responsible for C4b binding. We have completed the backbone and side-chain resonance assignments of the 11.3kDa fourth domain of Eap. The assignment data is published in the Journal of Biomolecular NMR Assignments. Further work on tertiary structure determination is under progress.

Publications

Type: Journal Articles Status: Published Year Published: 2016 Citation: Herrera, A.; Tomich, J., and Prakash, O. (2016) Membrane Active Peptides. Current Protein and Peptide Science 17:827-841.
Type: Journal Articles Status: Published Year Published: 2016 Citation: Woehl, J., Takahashi, D., Herrera, A., Anbanandam, A., Geisbrecht and Prakash, O. (2016) Backbone and side-chain 1H, 15N, and 13C resonance assignments of Extracellular Adherence Protein Domain 4. Biomol. NMR Assign. 10:301-305.

Progress 10/01/14 to 09/30/15

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: Mr. Lynn Schrag, Graduate Student Mr. Jordan Woehl, Graduate student Mr. Alvaro Herrera, Graduate Student Mr. Qasim Al Souhail, Graduate Stident How have the results been disseminated to communities of interest?Research publications and an oral presentations at an international meeting. 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 stress responsive peptides, modified starch, 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 prepared grant proposals for submission to NIH shared instrumentation funding program. 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? In several moth species, proteolytic activation of plasmatocyte spreading peptide (PSP, also known as GBP for growth blocking peptide) stimulates hemocyte encapsulation. Homologs of GBP, including upstream ENF peptides (uENFs) and stress responsive peptides (SRPs) are identified in many insects including mosquitoes. Since most of these peptides are activated during stress conditions, such as wounding, infection, ligation, heat or cold shock, we choose SRP as a general term to describe these structurally related molecules. Besides growth blocking, AMP induction, hemocyte spreading and chemotaxis, SRPs play roles in brain morphogenesis and thanatosis. Stress responsive peptides are highly conserved in amino acid sequence, and are approximately 29 residues long with one disulfide bond. To better understand the multiple functions of Manduca SRP2 and its structure-function relationship, we have recently initiated a two-dimensional proton-proton Nuclear Magnetic Resonance (NMR) spectroscopy studies to determine its solution structure. We have completed proton chemical shift assignments for this 29 amino-acids containing peptide using a set of 2D proton-proton through bond and through space correlation experiments. The structure calculations based on distance constraints obtained from 2D NMR experiments are under progress.

Publications

Type: Journal Articles Status: Published Year Published: 2015 Citation: Sukhankar, P., Whitaker, S., Garcia, M., Herrera, A.; Prakash, O., and Tomich, J. (2015). Thermally Induced Conformational Transitions in Nascent Branched Amphiphilic Peptide Capsules. Langmuir, 31, 2946-2955.
Type: Journal Articles Status: Published Year Published: 2015 Citation: Guragain, Y., Herrera, A., Vadlani, P., and Prakash, O. (2015) Lignins of Bioenergy Crops. Natural Products Communications, 10, 201-208.