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Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.
Natalia Sevillano; Evan M. Green; Jörg Votteler; Dong Young Kim; Xuefeng Ren; Bei Yang; Xi Liu; André Luiz Lourenço; James H. Hurley; Shauna Farr-Jones; John D. Gross; Yifan Cheng; Charles S. Craik. Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes. PLOS ONE 2021, 16, e0250318 .
AMA StyleNatalia Sevillano, Evan M. Green, Jörg Votteler, Dong Young Kim, Xuefeng Ren, Bei Yang, Xi Liu, André Luiz Lourenço, James H. Hurley, Shauna Farr-Jones, John D. Gross, Yifan Cheng, Charles S. Craik. Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes. PLOS ONE. 2021; 16 (5):e0250318.
Chicago/Turabian StyleNatalia Sevillano; Evan M. Green; Jörg Votteler; Dong Young Kim; Xuefeng Ren; Bei Yang; Xi Liu; André Luiz Lourenço; James H. Hurley; Shauna Farr-Jones; John D. Gross; Yifan Cheng; Charles S. Craik. 2021. "Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes." PLOS ONE 16, no. 5: e0250318.
Botulinum neurotoxins (BoNT) are extremely potent and can induce respiratory failure, requiring long-term intensive care to prevent death. Recombinant monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics. In contrast, equine antitoxin cannot be used prophylactically and has a short half-life. Two three-mAb combinations are in development that specifically neutralize BoNT serotype A (BoNT/A) and B (BoNT/B). The three-mAb combinations addressing a single serotype provided pre-exposure prophylaxis in the guinea pig inhalation model. A lyophilized co-formulation of six mAbs, designated G03-52-01, that addresses both A and B serotypes is in development. Here, we investigated the efficacy of G03-52-01 to protect guinea pigs against an aerosol exposure challenge of BoNT/A1 or BoNT/B1. Previously, it was found that each antibody demonstrated a dose-dependent exposure and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intravenous (IV) injection. Here we show that G03-52-01, in a single IM injection of G03-52-01 administered 48 h pre-exposure, protected guinea pigs against an aerosol challenge of up to 238 LD50s of BoNT/A1 and 191 LD50s of BoNT/B1. These data suggest that a single IM administration of G03-52-01 provides pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A1 or BoNT/B1.
Doris Snow; Ronald Cobb; Juan Martinez; Isaac Finger-Baker; Laura Collins; Sara Terpening; Emily Syar; Nancy Niemuth; Dean Kobs; Roy Barnewall; Shauna Farr-Jones; James Marks; Milan Tomic. A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model. Toxins 2021, 13, 31 .
AMA StyleDoris Snow, Ronald Cobb, Juan Martinez, Isaac Finger-Baker, Laura Collins, Sara Terpening, Emily Syar, Nancy Niemuth, Dean Kobs, Roy Barnewall, Shauna Farr-Jones, James Marks, Milan Tomic. A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model. Toxins. 2021; 13 (1):31.
Chicago/Turabian StyleDoris Snow; Ronald Cobb; Juan Martinez; Isaac Finger-Baker; Laura Collins; Sara Terpening; Emily Syar; Nancy Niemuth; Dean Kobs; Roy Barnewall; Shauna Farr-Jones; James Marks; Milan Tomic. 2021. "A Monoclonal Antibody Combination against both Serotypes A and B Botulinum Toxin Prevents Inhalational Botulism in a Guinea Pig Model." Toxins 13, no. 1: 31.
A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins—MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.
Efrat T. Harel; Penelope M. Drake; Robyn M. Barfield; Irene Lui; Shauna Farr-Jones; Laura Van’T Veer; Zev J. Gartner; Evan M. Green; André Luiz Lourenço; Yifan Cheng; Byron C. Hann; David Rabuka; Charles S. Craik. Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer. Antibodies 2019, 8, 54 .
AMA StyleEfrat T. Harel, Penelope M. Drake, Robyn M. Barfield, Irene Lui, Shauna Farr-Jones, Laura Van’T Veer, Zev J. Gartner, Evan M. Green, André Luiz Lourenço, Yifan Cheng, Byron C. Hann, David Rabuka, Charles S. Craik. Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer. Antibodies. 2019; 8 (4):54.
Chicago/Turabian StyleEfrat T. Harel; Penelope M. Drake; Robyn M. Barfield; Irene Lui; Shauna Farr-Jones; Laura Van’T Veer; Zev J. Gartner; Evan M. Green; André Luiz Lourenço; Yifan Cheng; Byron C. Hann; David Rabuka; Charles S. Craik. 2019. "Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer." Antibodies 8, no. 4: 54.
Botulinum neurotoxins (BoNT) are potential biothreat agents due to their high lethality, potency, and ease of distribution, thus the development of antitoxins is a high priority to the US government. This study examined pre-clinical pharmacokinetic studies in rats of four oligoclonal anti-BoNT mAb-based therapeutics (NTM-1631, NTM-1632, NTM-1633, NTM-1634) for five BoNT serotypes (A, B, E, C, and D). NTM-1631, NTM-1632, and NTM-1633 each consist of three IgG1 mAbs, each with a distinct human or humanized variable region which bind to distinct epitopes on BoNT serotype A, B, or E respectively. NTM-1634 consists of four human immunoglobulin G1 (IgG1) mAbs binding BoNT C/D mosaic toxins. The mechanism of these antitoxins requires that three antibodies simultaneously bind toxin to achieve rapid clearance. Rats (total 378) displayed no adverse clinical signs attributed to antibody treatment from any of the antitoxins. Pharmacokinetic evaluation demonstrated that the individual mAbs are slowly eliminated, exhibiting dose-dependent exposure and long elimination half-lives ranging from 6.5 days to 10 days. There were no consistent differences observed between males and females or among the individual antibodies in each formulation in half-life. Anti-drug antibodies (ADA) were observed, as expected for human antibodies administered to rats. The results presented were used to support the clinical investigation of antibody-based botulism antitoxins.
Yero Espinoza; David Wong; Ago Ahene; Kenneth Der; Zachary Martinez; John Pham; Ronald R. Cobb; Shauna Farr-Jones; James. D. Marks; Milan T. Tomic. Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats. Toxins 2019, 11, 345 .
AMA StyleYero Espinoza, David Wong, Ago Ahene, Kenneth Der, Zachary Martinez, John Pham, Ronald R. Cobb, Shauna Farr-Jones, James. D. Marks, Milan T. Tomic. Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats. Toxins. 2019; 11 (6):345.
Chicago/Turabian StyleYero Espinoza; David Wong; Ago Ahene; Kenneth Der; Zachary Martinez; John Pham; Ronald R. Cobb; Shauna Farr-Jones; James. D. Marks; Milan T. Tomic. 2019. "Pharmacokinetics of Human Recombinant Anti-Botulinum Toxin Antibodies in Rats." Toxins 11, no. 6: 345.
Botulinum neurotoxins (BoNT) are some of the most toxic proteins known, with a human LD50 of ~1 ng/kg. Equine antitoxin has a half-life in circulation of less than 1 day and is limited to a treatment rather than a prevention indication. The development of monoclonal antibodies (mAbs) may represent an alternative therapeutic option that can be produced at high quantities and of high quality and with half-lives of >10 days. Two different three mAb combinations are being developed that specifically neutralize BoNT serotypes A (BoNT/A) and B (BoNT/B). We investigated the pharmacokinetics of the anti-BoNT/A and anti-BoNT/B antibodies in guinea pigs (Cavia porcellus) and their ability to protect guinea pigs against an aerosol challenge of BoNT/A1 or BoNT/B1. Each antibody exhibited dose-dependent exposure and reached maximum circulating concentrations within 48 h post intraperitoneal or intramuscular injection. A single intramuscular dose of the three mAb combination protected guinea pigs against an aerosol challenge dose of 93 LD50 of BoNT/A1 and 116 LD50 of BoNT/B1 at 48 h post antibody administration. These mAbs are effective in preventing botulism after an aerosol challenge of BoNT/A1 and BoNT/B1 and may represent an alternative to vaccination to prevent type A or B botulism in those at risk of BoNT exposure.
Milan T. Tomic; Yero Espinoza; Zachary Martinez; Khanh Pham; Ronald R. Cobb; Doris M. Snow; Christopher G. Earnhart; Traci Pals; Emily S. Syar; Nancy Niemuth; Dean J. Kobs; Shauna Farr-Jones; James D. Marks. Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model. Toxins 2019, 11, 208 .
AMA StyleMilan T. Tomic, Yero Espinoza, Zachary Martinez, Khanh Pham, Ronald R. Cobb, Doris M. Snow, Christopher G. Earnhart, Traci Pals, Emily S. Syar, Nancy Niemuth, Dean J. Kobs, Shauna Farr-Jones, James D. Marks. Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model. Toxins. 2019; 11 (4):208.
Chicago/Turabian StyleMilan T. Tomic; Yero Espinoza; Zachary Martinez; Khanh Pham; Ronald R. Cobb; Doris M. Snow; Christopher G. Earnhart; Traci Pals; Emily S. Syar; Nancy Niemuth; Dean J. Kobs; Shauna Farr-Jones; James D. Marks. 2019. "Monoclonal Antibody Combinations Prevent Serotype A and Serotype B Inhalational Botulism in a Guinea Pig Model." Toxins 11, no. 4: 208.
Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.
Sujeewa D. Wijesuriya; Elizabeth Pongo; Milan Tomic; Fangjiu Zhang; Consuelo Garcia-Rodriquez; Fraser Conrad; Shauna Farr-Jones; James D. Marks; Arnold H. Horwitz. Antibody engineering to improve manufacturability. Protein Expression and Purification 2018, 149, 75 -83.
AMA StyleSujeewa D. Wijesuriya, Elizabeth Pongo, Milan Tomic, Fangjiu Zhang, Consuelo Garcia-Rodriquez, Fraser Conrad, Shauna Farr-Jones, James D. Marks, Arnold H. Horwitz. Antibody engineering to improve manufacturability. Protein Expression and Purification. 2018; 149 ():75-83.
Chicago/Turabian StyleSujeewa D. Wijesuriya; Elizabeth Pongo; Milan Tomic; Fangjiu Zhang; Consuelo Garcia-Rodriquez; Fraser Conrad; Shauna Farr-Jones; James D. Marks; Arnold H. Horwitz. 2018. "Antibody engineering to improve manufacturability." Protein Expression and Purification 149, no. : 75-83.
Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration. The lyophilized powder formulation contains nine mAbs, three mAbs for each of the three serotypes (A, B, E). The formulation was stored as a liquid and lyophilized powder for up to one year, and characterized by binding affinity and multiple physicochemical methods. No significant increase in soluble higher order aggregates, cleavage products, or change in charge isoforms was measured after storage as a lyophilized powder at 50°C for one year. Furthermore, toxin-domain binding ELISA data indicated that each of the individual antibodies in the lyophilized drug product showed essentially full binding capability to their respective toxin domains after being stored at 50°C for one year. Physicochemical characterization of the formulation demonstrated the nine individual mAbs were remarkably stable. This work demonstrates feasibility of lyophilized, oligoclonal antibody therapies for biodefense with ambient temperature stability, that would facilitate stockpiling, distribution, and administration.
Mingxiang Li; Dennis Lee; Chidi R. Obi; Joel K. Freeberg; Shauna Farr-Jones; Milan T. Tomic. An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E. PLoS ONE 2018, 13, e0197011 .
AMA StyleMingxiang Li, Dennis Lee, Chidi R. Obi, Joel K. Freeberg, Shauna Farr-Jones, Milan T. Tomic. An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E. PLoS ONE. 2018; 13 (5):e0197011.
Chicago/Turabian StyleMingxiang Li; Dennis Lee; Chidi R. Obi; Joel K. Freeberg; Shauna Farr-Jones; Milan T. Tomic. 2018. "An ambient temperature-stable antitoxin of nine co-formulated antibodies for botulism caused by serotypes A, B and E." PLoS ONE 13, no. 5: e0197011.
Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.
Consuelo Garcia-Rodriguez; Ali Razai; Isin Geren; Jianlong Lou; Fraser Conrad; Wei-Hua Wen; Shauna Farr-Jones; Theresa J. Smith; Jennifer L. Brown; Janet C. Skerry; Leonard A. Smith; James D. Marks. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes. Toxins 2018, 10, 105 .
AMA StyleConsuelo Garcia-Rodriguez, Ali Razai, Isin Geren, Jianlong Lou, Fraser Conrad, Wei-Hua Wen, Shauna Farr-Jones, Theresa J. Smith, Jennifer L. Brown, Janet C. Skerry, Leonard A. Smith, James D. Marks. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes. Toxins. 2018; 10 (3):105.
Chicago/Turabian StyleConsuelo Garcia-Rodriguez; Ali Razai; Isin Geren; Jianlong Lou; Fraser Conrad; Wei-Hua Wen; Shauna Farr-Jones; Theresa J. Smith; Jennifer L. Brown; Janet C. Skerry; Leonard A. Smith; James D. Marks. 2018. "A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes." Toxins 10, no. 3: 105.
The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.
Jianlong Lou; Weihua Wen; Fraser Conrad; Qi Meng; Jianbo Dong; Zhengda Sun; Consuelo Garcia-Rodriguez; Shauna Farr-Jones; Luisa W. Cheng; Thomas D. Henderson; Jennifer L. Brown; Theresa J. Smith; Leonard A. Smith; Anthony Cormier; James D. Marks. A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A. Toxins 2018, 10, 84 .
AMA StyleJianlong Lou, Weihua Wen, Fraser Conrad, Qi Meng, Jianbo Dong, Zhengda Sun, Consuelo Garcia-Rodriguez, Shauna Farr-Jones, Luisa W. Cheng, Thomas D. Henderson, Jennifer L. Brown, Theresa J. Smith, Leonard A. Smith, Anthony Cormier, James D. Marks. A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A. Toxins. 2018; 10 (2):84.
Chicago/Turabian StyleJianlong Lou; Weihua Wen; Fraser Conrad; Qi Meng; Jianbo Dong; Zhengda Sun; Consuelo Garcia-Rodriguez; Shauna Farr-Jones; Luisa W. Cheng; Thomas D. Henderson; Jennifer L. Brown; Theresa J. Smith; Leonard A. Smith; Anthony Cormier; James D. Marks. 2018. "A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A." Toxins 10, no. 2: 84.
Anwer Mujeeb; Nikolai B. Ulyanov; Todd M. Billeci; Shauna Farr-Jones; Thomas L. James. Conformational Ensemble Calculations: Analysis of Protein and Nucleic Acid NMR Data. Biological Magnetic Resonance 2006, 17, 201 -222.
AMA StyleAnwer Mujeeb, Nikolai B. Ulyanov, Todd M. Billeci, Shauna Farr-Jones, Thomas L. James. Conformational Ensemble Calculations: Analysis of Protein and Nucleic Acid NMR Data. Biological Magnetic Resonance. 2006; 17 ():201-222.
Chicago/Turabian StyleAnwer Mujeeb; Nikolai B. Ulyanov; Todd M. Billeci; Shauna Farr-Jones; Thomas L. James. 2006. "Conformational Ensemble Calculations: Analysis of Protein and Nucleic Acid NMR Data." Biological Magnetic Resonance 17, no. : 201-222.
Sandra Fox; Helen Wang; Lynne Sopchak; Shauna Farr-Jones. High Throughput Screening 2002: Moving Toward Increased Success Rates. Journal of Biomolecular Screening 2002, 7, 313 -316.
AMA StyleSandra Fox, Helen Wang, Lynne Sopchak, Shauna Farr-Jones. High Throughput Screening 2002: Moving Toward Increased Success Rates. Journal of Biomolecular Screening. 2002; 7 (4):313-316.
Chicago/Turabian StyleSandra Fox; Helen Wang; Lynne Sopchak; Shauna Farr-Jones. 2002. "High Throughput Screening 2002: Moving Toward Increased Success Rates." Journal of Biomolecular Screening 7, no. 4: 313-316.
Daina Avizonis; Shauna Farr-Jones. The Internet for nuclear magnetic resonance spectroscopists. Methods in Enzymology 2002, 338, 247 -259e.
AMA StyleDaina Avizonis, Shauna Farr-Jones. The Internet for nuclear magnetic resonance spectroscopists. Methods in Enzymology. 2002; 338 ():247-259e.
Chicago/Turabian StyleDaina Avizonis; Shauna Farr-Jones. 2002. "The Internet for nuclear magnetic resonance spectroscopists." Methods in Enzymology 338, no. : 247-259e.
NMR has been used to refine the structure of Syrian hamster (SHa) prion protein rPrP(90-231), which is commensurate with the infectious protease-resistant core of the scrapie prion protein PrPSc. The structure of rPrP(90-231), refolded to resemble the normal cellular isoform PrPC spectroscopically and immunologically, has been studied using multidimensional NMR; initial results were published [James et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10086-10091]. We now report refinement with better definition revealing important structural and dynamic features which can be related to biological observations pertinent to prion diseases. Structure refinement was based on 2778 unambiguously assigned nuclear Overhauser effect (NOE) connectivities, 297 ambiguous NOE restraints, and 63 scalar coupling constants (3JHNHa). The structure is represented by an ensemble of 25 best-scoring structures from 100 structures calculated using ARIA/X-PLOR and further refined with restrained molecular dynamics using the AMBER 4.1 force field with an explicit shell of water molecules. The rPrP(90-231) structure features a core domain (residues 125-228), with a backbone atomic root-mean-square deviation (RMSD) of 0.67 A, consisting of three alpha-helices (residues 144-154, 172-193, and 200-227) and two short antiparallel beta-strands (residues 129-131 and 161-163). The N-terminus (residues 90-119) is largely unstructured despite some sparse and weak medium-range NOEs implying the existence of bends or turns. The transition region between the core domain and flexible N-terminus, i.e., residues 113-128, consists of hydrophobic residues or glycines and does not adopt any regular secondary structure in aqueous solution. There are about 30 medium- and long-range NOEs within this hydrophobic cluster, so it clearly manifests structure. Multiple discrete conformations are evident, implying the possible existence of one or more metastable states, which may feature in conversion of PrPC to PrPSc. To obtain a more comprehensive picture of rPrP(90-231), dynamics have been studied using amide hydrogen-deuterium exchange and 15N NMR relaxation times (T1 and T2) and 15N{1H} NOE measurements. Comparison of the structure with previous reports suggests sequence-dependent features that may be reflected in a species barrier to prion disease transmission.
He Liu; Shauna Farr-Jones; Nikolai B. Ulyanov; Manuel Llinás; Susan Marqusee; Darlene Groth; Fred E. Cohen; Stanley B. Prusiner; Thomas L. James. Solution Structure of Syrian Hamster Prion Protein rPrP(90−231)†. Biochemistry 1999, 38, 5362 -5377.
AMA StyleHe Liu, Shauna Farr-Jones, Nikolai B. Ulyanov, Manuel Llinás, Susan Marqusee, Darlene Groth, Fred E. Cohen, Stanley B. Prusiner, Thomas L. James. Solution Structure of Syrian Hamster Prion Protein rPrP(90−231)†. Biochemistry. 1999; 38 (17):5362-5377.
Chicago/Turabian StyleHe Liu; Shauna Farr-Jones; Nikolai B. Ulyanov; Manuel Llinás; Susan Marqusee; Darlene Groth; Fred E. Cohen; Stanley B. Prusiner; Thomas L. James. 1999. "Solution Structure of Syrian Hamster Prion Protein rPrP(90−231)†." Biochemistry 38, no. 17: 5362-5377.
A method called PARSE (Probability Assessment via Relaxation rates of a Structural Ensemble) is described for determination of ensembles of structures from NMR data. The problem is approached in two separate steps: (1) generation of a pool of potential conformers, and (2) determination of the conformers' probabilities which best account for the experimental data. The probabilities are calculated by a global constrained optimization of a quadratic objective function measuring the agreement between observed NMR parameters and those calculated for the ensemble. The performance of the method is tested on synthetic data sets simulated for various structural ensembles of the complementary dinucleotide d(CA)·d(TG)
Nikolai B. Ulyanov; Anwer Mujeeb; Alessandro Donati; Patrick Furrer; He Liu; Shauna Farr-Jones; David E. Konerding; Uli Schmitz; Thomas L. James. Determination of Structural Ensembles from NMR Data: Conformational Sampling and Probability Assessment. ACS Symposium Series 1997, 181 -194.
AMA StyleNikolai B. Ulyanov, Anwer Mujeeb, Alessandro Donati, Patrick Furrer, He Liu, Shauna Farr-Jones, David E. Konerding, Uli Schmitz, Thomas L. James. Determination of Structural Ensembles from NMR Data: Conformational Sampling and Probability Assessment. ACS Symposium Series. 1997; ():181-194.
Chicago/Turabian StyleNikolai B. Ulyanov; Anwer Mujeeb; Alessandro Donati; Patrick Furrer; He Liu; Shauna Farr-Jones; David E. Konerding; Uli Schmitz; Thomas L. James. 1997. "Determination of Structural Ensembles from NMR Data: Conformational Sampling and Probability Assessment." ACS Symposium Series , no. : 181-194.
Daina Z. Avizonis; Shauna Farr-Jones; Phyllis Anne Kosen; Vladimir J. Basus. Conformations and Dynamics of the Essential Cysteinyl-Cysteine Ring Derived from the Acetylcholine Receptor†,‡. Journal of the American Chemical Society 1996, 118, 13031 -13039.
AMA StyleDaina Z. Avizonis, Shauna Farr-Jones, Phyllis Anne Kosen, Vladimir J. Basus. Conformations and Dynamics of the Essential Cysteinyl-Cysteine Ring Derived from the Acetylcholine Receptor†,‡. Journal of the American Chemical Society. 1996; 118 (51):13031-13039.
Chicago/Turabian StyleDaina Z. Avizonis; Shauna Farr-Jones; Phyllis Anne Kosen; Vladimir J. Basus. 1996. "Conformations and Dynamics of the Essential Cysteinyl-Cysteine Ring Derived from the Acetylcholine Receptor†,‡." Journal of the American Chemical Society 118, no. 51: 13031-13039.
We have determined the solution structure of the omega-conotoxin MVIIC from Conus magus by 1H NMR. This conopeptide preferentially blocks P and Q type Ca2+ currents by binding with high affinity to voltage-sensitive Ca2+ channels in neurons. This 26 residue peptide with three disulfide bonds was chemically synthesized and refolded for NMR structural studies. The 1H NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the beta prochiral centers. Complete relaxation matrix analysis using MARDIGRAS was used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined by measuring 13C relaxation times using inversely detected natural abundance spectra. Distances were input to DG, which provided 15 starting structures which were then subjected to restrained molecular dynamics calculations using SANDER with the AMBER 91 force field in vacuo. 1H-1H vicinal coupling constants were obtained using a combination of line fitting of both E. COSY and NOESY spectra and used to generate angle restraints that were included explicitly in the restrained molecular dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 A. The ensemble R1/6 factor calculated by CORMA for the final 15 structures was 11%. The final structure consists of an anti-parallel, triple-stranded beta-sheet, with four turns. In spite of significant differences in amino acid sequence and affinities for calcium channel subtypes, the backbone structure of omega-conotoxin MVIIC is very similar to the previously reported structure of omega-conotoxin GVIA.
Shauna Farr-Jones; George P. Miljanich; Laszlo Nadasdi; J. Ramachandran; Vladimir J. Basus. Solution Structure of ω-Conotoxin MVIIC, a High Affinity Ligand of P-type Calcium Channels, using1H NMR Spectroscopy and Complete Relaxation Matrix Analysis. Journal of Molecular Biology 1995, 248, 106 -124.
AMA StyleShauna Farr-Jones, George P. Miljanich, Laszlo Nadasdi, J. Ramachandran, Vladimir J. Basus. Solution Structure of ω-Conotoxin MVIIC, a High Affinity Ligand of P-type Calcium Channels, using1H NMR Spectroscopy and Complete Relaxation Matrix Analysis. Journal of Molecular Biology. 1995; 248 (1):106-124.
Chicago/Turabian StyleShauna Farr-Jones; George P. Miljanich; Laszlo Nadasdi; J. Ramachandran; Vladimir J. Basus. 1995. "Solution Structure of ω-Conotoxin MVIIC, a High Affinity Ligand of P-type Calcium Channels, using1H NMR Spectroscopy and Complete Relaxation Matrix Analysis." Journal of Molecular Biology 248, no. 1: 106-124.
Rat trypsin II has been converted to a protease with chymotrypsin-like substrate specificity [Hedstrom, L., et al. (1994) Biochemistry (preceding paper in this issue)]. The key alteration in this conversion is the exchange of two surface loops for the analogous loops of chymotrypsin. k(inact)/Ki for the inactivation of chymotrypsin, trypsin, a trypsin mutant with poor activity (D189S), and the chymotrypsin-like mutants Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] by Suc-Ala-Ala-Pro-Phe-chloromethylketone correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. k(inact)'s for the inactivation of Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] are comparable to that of chymotrypsin, while Ki's were much higher. Ki for the inhibition of these enzymes by the transition-state analog MeOSuc-Ala-Ala-Pro-boro-Phe also correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. These results suggest that the surface loops stabilize the transition state for hydrolysis of chymotrypsin substrates by improving the orientation of bound substrates relative to the catalytic residues. Lastly, trypsin and chymotrypsin have comparable affinities for proflavin, while the Kd for the Tr-->Ch[S1+L1+L2+Y172W]-proflavin complex is 10-fold higher. No proflavin binding could be observed for either D189S or Tr-->Ch-[S1+L1+L2], which suggests that the S1 binding pockets of these two mutant enzymes are deformed. This work confirms that enzyme specificity is expressed in the chemical steps of the reaction rather than in substrate binding.
Lizbeth Hedstrom; Shauna Farr-Jones; Charles A. Kettner; William J. Rutter. Converting Trypsin to Chymotrypsin: Ground-State Binding Does Not Determine Substrate Specificity. Biochemistry 1994, 33, 8764 -8769.
AMA StyleLizbeth Hedstrom, Shauna Farr-Jones, Charles A. Kettner, William J. Rutter. Converting Trypsin to Chymotrypsin: Ground-State Binding Does Not Determine Substrate Specificity. Biochemistry. 1994; 33 (29):8764-8769.
Chicago/Turabian StyleLizbeth Hedstrom; Shauna Farr-Jones; Charles A. Kettner; William J. Rutter. 1994. "Converting Trypsin to Chymotrypsin: Ground-State Binding Does Not Determine Substrate Specificity." Biochemistry 33, no. 29: 8764-8769.
Shauna Farr-Jones; Winona Y. L. Wong; William Gutheil; William W. Bachovchin. Direct observation of the tautomeric forms of histidine in nitrogen-15 NMR spectra at low temperatures. Comments on intramolecular hydrogen bonding and on tautomeric equilibrium constants. Journal of the American Chemical Society 1993, 115, 6813 -6819.
AMA StyleShauna Farr-Jones, Winona Y. L. Wong, William Gutheil, William W. Bachovchin. Direct observation of the tautomeric forms of histidine in nitrogen-15 NMR spectra at low temperatures. Comments on intramolecular hydrogen bonding and on tautomeric equilibrium constants. Journal of the American Chemical Society. 1993; 115 (15):6813-6819.
Chicago/Turabian StyleShauna Farr-Jones; Winona Y. L. Wong; William Gutheil; William W. Bachovchin. 1993. "Direct observation of the tautomeric forms of histidine in nitrogen-15 NMR spectra at low temperatures. Comments on intramolecular hydrogen bonding and on tautomeric equilibrium constants." Journal of the American Chemical Society 115, no. 15: 6813-6819.
15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.
William W. Bachovchin; Winona Y. L. Wong; Shauna Farr-Jones; Ashok B. Shenvi; Charles A. Kettner. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes. Biochemistry 1988, 27, 7689 -7697.
AMA StyleWilliam W. Bachovchin, Winona Y. L. Wong, Shauna Farr-Jones, Ashok B. Shenvi, Charles A. Kettner. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes. Biochemistry. 1988; 27 (20):7689-7697.
Chicago/Turabian StyleWilliam W. Bachovchin; Winona Y. L. Wong; Shauna Farr-Jones; Ashok B. Shenvi; Charles A. Kettner. 1988. "Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes." Biochemistry 27, no. 20: 7689-7697.