10 results on '"Jason C. Klima"'
Search Results
2. Incorporation of sensing modalities into de novo designed fluorescence-activating proteins
- Author
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Jason C. Klima, Lindsey A. Doyle, Justin Daho Lee, Michael Rappleye, Lauren A. Gagnon, Min Yen Lee, Emilia P. Barros, Anastassia A. Vorobieva, Jiayi Dou, Samantha Bremner, Jacob S. Quon, Cameron M. Chow, Lauren Carter, David L. Mack, Rommie E. Amaro, Joshua C. Vaughan, Andre Berndt, Barry L. Stoddard, and David Baker
- Subjects
Science - Abstract
Fluorescent protein reporters based on GFP exist, but have intrinsic disadvantages. Here the authors incorporate pH, Ca2+ and protein–protein interaction sensing modalities into de novo designed mini-fluorescence-activating proteins (mFAPs), with increased photostability and smaller size, which bind a range of DFHBI chromophore variants.
- Published
- 2021
- Full Text
- View/download PDF
3. Inhibition of a malaria host–pathogen interaction by a computationally designed inhibitor
- Author
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Autumn R. Tobin, Rachel Crow, Darya V. Urusova, Jason C. Klima, Niraj H. Tolia, and Eva‐Maria Strauch
- Subjects
Molecular Biology ,Biochemistry - Abstract
Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/Receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria. This article is protected by copyright. All rights reserved.
- Published
- 2022
- Full Text
- View/download PDF
4. PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design
- Author
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Morgan L. Nance, Steven J. Bertolani, William R. Schief, Rebecca F. Alford, Daniel W. Kulp, Jason C. Klima, Shourya S. Roy Burman, Yuanhan Wu, Jack Maguire, Jordan R. Willis, Roland L. Dunbrack, Andrew Leaver-Fay, Jason W. Labonte, Aleexsan Adal, Ramya Rangan, Brian Kuhlman, Sergey Lyskov, Jared Adolf-Bryfogle, Justin B. Siegel, Kathy H. Le, Rhiju Das, Jeffrey J. Gray, Brian D. Weitzner, and Matt A. Adrianowycz
- Subjects
0303 health sciences ,03 medical and health sciences ,0302 clinical medicine ,Computer science ,Nanotechnology ,Biomolecular structure ,Article ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of 16 modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications, such as protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a Web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels, as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.
- Published
- 2021
- Full Text
- View/download PDF
5. Incorporation of sensing modalities into de novo designed fluorescence-activating proteins
- Author
-
Samantha Bremner, Lindsey Doyle, Andre Berndt, Jason C. Klima, David L. Mack, Jiayi Dou, Lauren Carter, Justin Daho Lee, Emilia P. Barros, Joshua C. Vaughan, Cameron M. Chow, David Baker, Min Yen Lee, Michael Rappleye, Rommie E. Amaro, Jacob S. Quon, Lauren A. Gagnon, Anastassia A. Vorobieva, Barry L. Stoddard, and Department of Bio-engineering Sciences
- Subjects
Models, Molecular ,0301 basic medicine ,Chemistry(all) ,Science ,Green Fluorescent Proteins ,Gfp reporter ,General Physics and Astronomy ,Bioengineering ,Physics and Astronomy(all) ,01 natural sciences ,Article ,Fluorescence ,General Biochemistry, Genetics and Molecular Biology ,Green fluorescent protein ,03 medical and health sciences ,Models ,Chlorocebus aethiops ,0103 physical sciences ,Animals ,Humans ,Fluorescent protein ,Acetylcholine metabolism ,X-ray crystallography ,Fluorescent Dyes ,Multidisciplinary ,010304 chemical physics ,Biochemistry, Genetics and Molecular Biology(all) ,Extramural ,Chemistry ,Molecular ,General Chemistry ,Fluorescent proteins ,Hydrogen-Ion Concentration ,Acetylcholine ,Luminescent Proteins ,Fluorescence intensity ,Wide-field fluorescence microscopy ,HEK293 Cells ,030104 developmental biology ,COS Cells ,Biophysics ,Calcium ,Generic health relevance ,Protein design - Abstract
Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering., Fluorescent protein reporters based on GFP exist, but have intrinsic disadvantages. Here the authors incorporate pH, Ca2+ and protein–protein interaction sensing modalities into de novo designed mini-fluorescence-activating proteins (mFAPs), with increased photostability and smaller size, which bind a range of DFHBI chromophore variants.
- Published
- 2021
6. Inhibition of a critical malaria host-pathogen interaction by a computationally designed inhibitor targeting Plasmodium vivax DBP
- Author
-
Autumn R Tobin, Rachel Crow, Darya V. Urusova, Jason C. Klima, Niraj H. Tolia, and Eva-Maria Strauch
- Subjects
parasitic diseases - Abstract
Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. To address this need, we developed nanomolar inhibitor that could serve as a therapeutic and a diagnostic. A unique characteristic of P. vivax is that it depends on the Duffy antigen/Receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.TeaserDe novo designed proteins present a new alternative for the development of therapeutics. They can be small, highly stable and easily manufactured. Here we designed a potential new therapeutic to inhibit entry of Plasmodium vivax into red blood cells by interfering its interactions of surface displayed DBP molecules with the host receptor DARC.
- Published
- 2022
- Full Text
- View/download PDF
7. Bacterial expression and protein purification of mini-fluorescence-activating proteins
- Author
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Cameron M. Chow, Jason C. Klima, Jiayi Dou, David Baker, Lauren Carter, and Anastassia A. Vorobieva
- Subjects
Text mining ,Biochemistry ,business.industry ,Chemistry ,Protein purification ,business ,Fluorescence - Abstract
Bacterial expression and purification of de novo designed mini-fluorescence-activating proteins (mFAPs) are accomplished by genetically fusing mFAP variants to an N-terminal 6xHis tag and optionally to a Tobacco Etch Virus (TEV) protease epitope. The protocol can take less than 24 hours to complete, and allows for downstream in vitro characterization of photophysical properties of mFAPs by equilibration with exogenous fluorogenic compounds.
- Published
- 2021
- Full Text
- View/download PDF
8. PyRosetta Jupyter Notebooks Teach Biomolecular Structure Prediction and Design
- Author
-
Jeffrey J. Gray, Jack Maguire, Steven J. Bertolani, Rhiju Das, Brian D. Weitzner, Ramya Rangan, Aleexan Adal, Kathy H. Le, Morgan L. Nance, Jason C. Klima, Jared Adolf-Bryfogle, Brian Kuhlman, Matt A. Adrianowycz, Jason W. Labonte, Sergey Lyskov, Andrew Leaver-Fay, Shourya S. Roy Burman, Roland L. Dunbrack, William R. Schief, Rebecca F. Alford, and Justin B. Siegel
- Subjects
Computer science ,0103 physical sciences ,05 social sciences ,050301 education ,Nanotechnology ,biomedical_chemical_engineering ,Biomolecular structure ,0503 education ,01 natural sciences ,010305 fluids & plasmas - Abstract
Biomolecular structure drives function, and computational capabilities have progressed such that the prediction and computational design of biomolecular structures is increasingly feasible. Because computational biophysics attracts students from many different backgrounds and with different levels of resources, teaching the subject can be challenging. One strategy to teach diverse learners is with interactive multimedia material that promotes self-paced, active learning. We have created a hands-on education strategy with a set of fifteen modules that teach topics in biomolecular structure and design, from fundamentals of conformational sampling and energy evaluation to applications like protein docking, antibody design, and RNA structure prediction. Our modules are based on PyRosetta, a Python library that encapsulates all computational modules and methods in the Rosetta software package. The workshop-style modules are implemented as Jupyter Notebooks that can be executed in the Google Colaboratory, allowing learners access with just a web browser. The digital format of Jupyter Notebooks allows us to embed images, molecular visualization movies, and interactive coding exercises. This multimodal approach may better reach students from different disciplines and experience levels as well as attract more researchers from smaller labs and cognate backgrounds to leverage PyRosetta in their science and engineering research. All materials are freely available at https://github.com/RosettaCommons/PyRosetta.notebooks.
- Published
- 2020
9. De novo design of tunable, pH-driven conformational changes
- Author
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Vicki H. Wysocki, Aniruddha Sahasrabuddhe, David Baker, Kelly K. Lee, Banumathi Sankaran, Zibo Chen, Kathy Y. Wei, Mengxuan Jia, Scott E. Boyken, Alexander Mileant, Carl Walkey, Florian Busch, Edgar A. Hodge, Neil P. King, Alfredo Quijano-Rubio, Mark A. Benhaim, Heejun Choi, Sarah Byron, Jason C. Klima, Jennifer Lippincott-Schwartz, and Matthew J. Bick
- Subjects
Multidisciplinary ,General Science & Technology ,Chemistry ,Protein Conformation ,Protein Stability ,Protein design ,Protonation ,Cooperativity ,Hydrogen-Ion Concentration ,Protein Engineering ,Article ,Steric repulsion ,Hydrophobic effect ,Membrane ,Protein structure ,Biophysics ,Generic health relevance ,sense organs ,Protein Multimerization ,Histidine - Abstract
The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.
- Published
- 2018
10. Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellateLingulodinium polyedrum: evidence for the role of stretch-activated ion channels
- Author
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Michael I. Latz, Kelly Jin, Grant B. Deane, M. D. Stokes, and Jason C. Klima
- Subjects
chemistry.chemical_element ,Plant Science ,Aquatic Science ,Biology ,Calcium ,biology.organism_classification ,Cell biology ,chemistry ,Membrane fluidity ,Bioluminescence ,Signal transduction ,Mechanotransduction ,Lingulodinium polyedrum ,Ion channel ,Intracellular - Abstract
Dinoflagellate bioluminescence serves as a whole-cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage-sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd(3+) ), a nonspecific blocker of stretch-activated and some voltage-gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L-type voltage-gated Ca(2+) channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol-stimulated bioluminescence was inhibited by Gd(3+) but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch-activated and voltage-gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch-activated component at the plasma membrane.
- Published
- 2013
- Full Text
- View/download PDF
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