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  • 1
    Publication Date: 2009-08-21
    Description: The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2766670/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2766670/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Wu, Yi I -- Frey, Daniel -- Lungu, Oana I -- Jaehrig, Angelika -- Schlichting, Ilme -- Kuhlman, Brian -- Hahn, Klaus M -- GM057464/GM/NIGMS NIH HHS/ -- GM64346/GM/NIGMS NIH HHS/ -- R01 GM057464/GM/NIGMS NIH HHS/ -- R01 GM057464-09/GM/NIGMS NIH HHS/ -- U54 GM064346/GM/NIGMS NIH HHS/ -- U54 GM064346-089026/GM/NIGMS NIH HHS/ -- England -- Nature. 2009 Sep 3;461(7260):104-8. doi: 10.1038/nature08241. Epub 2009 Aug 19.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA. yiwu@med.unc.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19693014" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Avena/genetics ; Cell Line ; *Cell Movement/radiation effects ; Cell Surface Extensions ; Cell Survival ; Cryptochromes ; Crystallization ; Crystallography, X-Ray ; Embryo, Mammalian/cytology ; Enzyme Activation/radiation effects ; Fibroblasts ; Flavoproteins/chemistry/genetics/metabolism ; Fluorescence Recovery After Photobleaching ; Genetic Engineering/*methods ; HeLa Cells ; Humans ; Mice ; Models, Molecular ; Myosins/metabolism ; Protein Conformation ; rac1 GTP-Binding Protein/chemistry/*genetics/*metabolism/radiation effects ; rho GTP-Binding Proteins/antagonists & inhibitors/metabolism
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
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    American Association for the Advancement of Science (AAAS)
    Publication Date: 2011-05-14
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Der, Bryan S -- Kuhlman, Brian -- New York, N.Y. -- Science. 2011 May 13;332(6031):801-2. doi: 10.1126/science.1207082.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA. bder@email.unc.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21566181" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; *Computer Simulation ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry/*metabolism ; Hydrophobic and Hydrophilic Interactions ; Models, Molecular ; Peptide Library ; Protein Binding ; Protein Conformation ; *Protein Engineering ; Proteins/*chemistry/genetics/*metabolism ; Software
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 3
    Publication Date: 2003-11-25
    Description: A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kuhlman, Brian -- Dantas, Gautam -- Ireton, Gregory C -- Varani, Gabriele -- Stoddard, Barry L -- Baker, David -- New York, N.Y. -- Science. 2003 Nov 21;302(5649):1364-8.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/14631033" target="_blank"〉PubMed〈/a〉
    Keywords: Algorithms ; Amino Acid Sequence ; Circular Dichroism ; Computational Biology ; Computer Graphics ; Computer Simulation ; Crystallization ; Crystallography, X-Ray ; Databases, Protein ; Models, Molecular ; Molecular Sequence Data ; Monte Carlo Method ; Nuclear Magnetic Resonance, Biomolecular ; *Protein Conformation ; Protein Denaturation ; *Protein Engineering ; *Protein Folding ; Protein Structure, Secondary ; Proteins/*chemistry ; *Software ; Solubility ; Temperature ; Thermodynamics
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2016-05-07
    Description: Natural recombination combines pieces of preexisting proteins to create new tertiary structures and functions. We describe a computational protocol, called SEWING, which is inspired by this process and builds new proteins from connected or disconnected pieces of existing structures. Helical proteins designed with SEWING contain structural features absent from other de novo designed proteins and, in some cases, remain folded at more than 100 degrees C. High-resolution structures of the designed proteins CA01 and DA05R1 were solved by x-ray crystallography (2.2 angstrom resolution) and nuclear magnetic resonance, respectively, and there was excellent agreement with the design models. This method provides a new strategy to rapidly create large numbers of diverse and designable protein scaffolds.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Jacobs, T M -- Williams, B -- Williams, T -- Xu, X -- Eletsky, A -- Federizon, J F -- Szyperski, T -- Kuhlman, B -- GM094597/GM/NIGMS NIH HHS/ -- R01GM073960/GM/NIGMS NIH HHS/ -- R01GM117968/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2016 May 6;352(6286):687-90. doi: 10.1126/science.aad8036.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Program in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. ; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. ; Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA. Northeast Structural Genomics Consortium. ; Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA. ; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. bkuhlman@email.unc.edu.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/27151863" target="_blank"〉PubMed〈/a〉
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2015-09-10
    Description: Dendritic spines are the major loci of synaptic plasticity and are considered as possible structural correlates of memory. Nonetheless, systematic manipulation of specific subsets of spines in the cortex has been unattainable, and thus, the link between spines and memory has been correlational. We developed a novel synaptic optoprobe, AS-PaRac1 (activated synapse targeting photoactivatable Rac1), that can label recently potentiated spines specifically, and induce the selective shrinkage of AS-PaRac1-containing spines. In vivo imaging of AS-PaRac1 revealed that a motor learning task induced substantial synaptic remodelling in a small subset of neurons. The acquired motor learning was disrupted by the optical shrinkage of the potentiated spines, whereas it was not affected by the identical manipulation of spines evoked by a distinct motor task in the same cortical region. Taken together, our results demonstrate that a newly acquired motor skill depends on the formation of a task-specific dense synaptic ensemble.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634641/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634641/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Hayashi-Takagi, Akiko -- Yagishita, Sho -- Nakamura, Mayumi -- Shirai, Fukutoshi -- Wu, Yi I -- Loshbaugh, Amanda L -- Kuhlman, Brian -- Hahn, Klaus M -- Kasai, Haruo -- GM102924/GM/NIGMS NIH HHS/ -- NS071216/NS/NINDS NIH HHS/ -- R01 GM102924/GM/NIGMS NIH HHS/ -- R21 NS071216/NS/NINDS NIH HHS/ -- England -- Nature. 2015 Sep 17;525(7569):333-8. doi: 10.1038/nature15257. Epub 2015 Sep 9.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033. ; PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. ; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. ; Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, Connecticut 06032, USA. ; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA. ; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA. ; Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26352471" target="_blank"〉PubMed〈/a〉
    Keywords: Animals ; Dendritic Spines/physiology/radiation effects ; Hippocampus/cytology/physiology/radiation effects ; In Vitro Techniques ; Light ; Long-Term Potentiation/physiology/radiation effects ; Male ; Memory/*physiology/*radiation effects ; Mice ; Molecular Probes ; Motor Cortex/cytology/*physiology/*radiation effects ; Motor Skills/physiology/radiation effects ; Neuronal Plasticity/*physiology/*radiation effects ; Rotarod Performance Test ; Spatio-Temporal Analysis ; Synapses/*physiology/*radiation effects
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 6
    Publication Date: 2010-10-25
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 7
    Publication Date: 2011-12-05
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 8
    Publication Date: 2001-08-28
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 9
    Publication Date: 2007-10-30
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 10
    Publication Date: 2000-09-12
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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