|
| ||
|
|
||
Bundle Protein (bundle + protein)
Selected AbstractsA Designed Well-Folded Monomeric Four-Helix Bundle Protein Prepared by Fmoc Solid-Phase Peptide Synthesis and Native Chemical Ligation,CHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2006Gunnar T. Dolphin Dr. Abstract The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide,thioester fragment was prepared by anchoring the free ,-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine,thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide,thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins. [source] Computational Design of Four-Helix Bundle Proteins That Bind Nonbiological CofactorsBIOTECHNOLOGY PROGRESS, Issue 1 2008Andreas Lehmann Recent work is discussed concerning the computational design of four-helix bundle proteins that form complexes with nonbiological cofactors. Given that often there are no suitable natural proteins to provide starting points in the creation of such nonbiological systems, computational design is well suited for the design and study of new protein-cofactor complexes. Recent design efforts are presented in the context of prior work on the de novo design and engineering of porphyrin-binding four-helix bundle proteins and current developments in nonlinear optical materials. Such protein-nonbiological cofactor complexes stand to enable new applications in protein science and materials research. [source] Inverse Monte Carlo procedure for conformation determination of macromoleculesJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 7 2003Mark Bathe Abstract A novel numerical method for determining the conformational structure of macromolecules is applied to idealized biomacromolecules in solution. The method computes effective inter-residue interaction potentials solely from the corresponding radial distribution functions, such as would be obtained from experimental data. The interaction potentials generate conformational ensembles that reproduce thermodynamic properties of the macromolecule (mean energy and heat capacity) in addition to the target radial distribution functions. As an evaluation of its utility in structure determination, we apply the method to a homopolymer and a heteropolymer model of a three-helix bundle protein [Zhou, Y.; Karplus, M. Proc Natl Acad Sci USA 1997, 94, 14429; Zhou, Y. et al. J Chem Phys 1997, 107, 10691] at various thermodynamic state points, including the ordered globule, disordered globule, and random coil states. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 876,890, 2003 [source] The kinetics of G-CSF foldingPROTEIN SCIENCE, Issue 10 2002David N. Brems Abstract The folding kinetics of G-CSF were determined by trp-fluorescence and far-UV circular dichroism. Folding and unfolding was achieved by rapid dilution and mixing of the denaturant, GdnHCl. G-CSF is a four-helical bundle protein with two long loops between the first and second helices and between the third and fourth helices. The entire conformational change expected by fluorescence was observed by stopped-flow technology, but due to rapid refolding kinetics only a portion was observed by circular dichroism. G-CSF contains two trp residues, and their contribution to the fluorescent-detected kinetics were deciphered through the use of single-site trp mutants. The trp moieties are probes of the local conformation surrounding their environment. One trp at residue 118 is located within the third helix while the other trp at residue 58 is part of the long loop between the first and second helices. The refolding results were most consistent with the following mechanism: U , I1 , I2 , N; where U represents the unfolded protein, I1 represents intermediate state 1, I2 represents intermediate state 2, and N represents the native state. I1 is characterized as having approximately one-half of the native-like helical structure and none of the native-like fluorescence. I2 has 100% of the native helical structure and most of the trp-118 and little of the trp-58 native-like fluorescence. Thus refolding occurs in distinct stages with half of the helix forming first followed by the remaining half of the helix including the third helix and finally the loop between the first and second helices folds. [source] Structure determination of a small protein through a 23-dimensional molecular-replacement searchACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2003Nicholas M. Glykos The crystal structure of a 4-,-helical bundle protein has been determined by the application of a 23-dimensional molecular-replacement search performed using a stochastic method. The search model for the calculation was a 26-residue-long polyalanine helix amounting to less than 13% of the total number of atoms in the asymmetric unit of the target crystal structure. The crystal structure determination procedure is presented in detail, with emphasis on the molecular-replacement calculations. [source] A Designed Well-Folded Monomeric Four-Helix Bundle Protein Prepared by Fmoc Solid-Phase Peptide Synthesis and Native Chemical Ligation,CHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2006Gunnar T. Dolphin Dr. Abstract The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide,thioester fragment was prepared by anchoring the free ,-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine,thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide,thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins. [source] Computational Design of Four-Helix Bundle Proteins That Bind Nonbiological CofactorsBIOTECHNOLOGY PROGRESS, Issue 1 2008Andreas Lehmann Recent work is discussed concerning the computational design of four-helix bundle proteins that form complexes with nonbiological cofactors. Given that often there are no suitable natural proteins to provide starting points in the creation of such nonbiological systems, computational design is well suited for the design and study of new protein-cofactor complexes. Recent design efforts are presented in the context of prior work on the de novo design and engineering of porphyrin-binding four-helix bundle proteins and current developments in nonlinear optical materials. Such protein-nonbiological cofactor complexes stand to enable new applications in protein science and materials research. [source] | ||||||||||