Sat, 05 Jul 2008 06:18:40 BST CiteULike: cactus's design http://www.citeulike.org/user/cactus/tag/design CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/cactus/article/2568656 <i>Nature (19 March 2008)</i> Kemp elimination catalysts by computational enzyme design Daniela Röthlisberger Olga Khersonsky Andrew Wollacott Lin Jiang Jason Dechancie Jamie Betker Jasmine Gallaher Eric Althoff Alexandre Zanghellini Orly Dym Shira Albeck Kendall Houk Dan Tawfik David Baker doi:10.1038/nature06879 Nature (19 March 2008) 2008-03-21T04:33:18-00:00 2008 Nature 0028-0836 Nature Publishing Group clip2 design enzyme protein http://www.citeulike.org/user/cactus/article/2368760 <i>Proceedings of the National Academy of Sciences, Vol. 105, No. 3. (22 January 2008), pp. 901-906.</i><br /><br />Many multifunctional tumor suppressor proteins have low stability, a property linked to cancer development. The von HippelLindau tumor suppressor protein (pVHL) is one of these proteins. pVHL forms part of the E3 ubiquitin ligase complex that regulates the degradation of the hypoxia-inducible factor (HIF). Under native conditions, free pVHL is a molten globule, but it is stabilized in the E3 complex. By using molecular dynamics simulations, we observed that the interface between the two pVHL domains is the least stable region in unbound pVHL. We designed five stable mutants: one with a mutation at the interdomain interface and the others in the alpha- or -domains. Experimentally, type 2B pVHL disease mutant Y98N at the HIF binding site was shown to destabilize pVHL and decrease its binding affinity to HIF. Our simulations showed that the decrease in pVHL stability and binding affinity are allosterically regulated. The mutations designed to stabilize unbound wild-type pVHL, which are away from the elongin C and HIF binding sites, successfully stabilized the Y98N pVHLelongin C complex and lowered the binding free energy of pVHL with HIF. Our results indicated both the enthalpic and dynamic allosteric components between the elongin C and HIF binding sites in pVHL, in the alpha- and -domains, respectively, mediated by the interdomain interface and linker. Drugs mimicking the allosteric effects of these mutants may rescue pVHL function in von HippelLindau disease. 10.1073/pnas.0707401105 Allosteric effects in the marginally stable von Hippel-Lindau tumor suppressor protein and allostery-based rescue mutant design Jin Liu Ruth Nussinov doi:10.1073/pnas.0707401105 Proceedings of the National Academy of Sciences, Vol. 105, No. 3. (22 January 2008), pp. 901-906. 2008-02-13T07:30:12-00:00 2008 Proceedings of the National Academy of Sciences 105 3 901 906 allostery design fullatom md mutation http://www.citeulike.org/user/cactus/article/2220255 <i>Science, Vol. 319, No. 5860. (11 January 2008), pp. 206-209.</i><br /><br />The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures. 10.1126/science.1150421 Designed Protein-Protein Association Dirk Grueninger Nora Treiber Mathias Ziegler Jochen Koetter Monika-Sarah Schulze Georg Schulz doi:10.1126/science.1150421 Science, Vol. 319, No. 5860. (11 January 2008), pp. 206-209. 2008-01-11T17:43:28-00:00 2008 Science 319 5860 206 209 clip2 design mutation oligomer protein http://www.citeulike.org/user/cactus/article/1942977 <i>Proceedings of the National Academy of Sciences, Vol. 104, No. 40. (2 October 2007), pp. 15677-15681.</i><br /><br />Proteinligand interactions, including proteinprotein interactions, are ubiquitously essential in biological processes and also have important applications in biotechnology. A wide range of methodologies have been developed for quantitative analysis of proteinligand interactions. However, most of them do not report direct functional/structural consequence of ligand binding. Instead they only detect the change of physical properties, such as fluorescence and refractive index, because of the colocalization of protein and ligand, and are susceptible to false positives. Thus, important information about the functional state of proteinligand complexes cannot be obtained directly. Here we report a functional single-molecule binding assay that uses force spectroscopy to directly probe the functional consequence of ligand binding and report the functional state of proteinligand complexes. As a proof of principle, we used protein G and the Fc fragment of IgG as a model system in this study. Binding of Fc to protein G does not induce major structural changes in protein G but results in significant enhancement of its mechanical stability. Using mechanical stability of protein G as an intrinsic functional reporter, we directly distinguished and quantified Fc-bound and Fc-free forms of protein G on a single-molecule basis and accurately determined their dissociation constant. This single-molecule functional binding assay is label-free, nearly background-free, and can detect functional heterogeneity, if any, among proteinligand interactions. This methodology opens up avenues for studying proteinligand interactions in a functional context, and we anticipate that it will find broad application in diverse proteinligand systems. 10.1073/pnas.0705367104 A functional single-molecule binding assay via force spectroscopy Yi Cao MM Balamurali Deepak Sharma Hongbin Li doi:10.1073/pnas.0705367104 Proceedings of the National Academy of Sciences, Vol. 104, No. 40. (2 October 2007), pp. 15677-15681. 2007-11-20T11:18:27-00:00 2007 Proceedings of the National Academy of Sciences 104 40 15677 15681 afm binding clip2 design experiment ligand protein