Dataset Information

Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels.

ABSTRACT: Automated methods for identifying and characterizing regular beta-barrels from coordinate data have been developed to analyze and classify various kinds of barrel structures based on geometric parameters such as the barrel strand number (n) and shear number (S). In total, we find 1,316 barrels in the January 1998 release of Protein Data Bank. Of 1,316 barrels, 1,277 barrels had an even shear number, corresponding to 50 nonhomologous families. The (beta alpha)8 triose phosphate isomerase (TIM) barrel (n = 8, S = 8) fold has the largest number of apparently nonhomologous entries, 16, although the trypsin like antiparallel (n = 6, S = 8) barrels (representing only three families) are the most common with 527 barrels. Of all the protein families that exhibit barrel structures, 68% are found to be various kinds of enzymes, the remainder being binding proteins and transport membrane proteins. In addition, the layers of side chains, which form the cores of barrels with S = n and S = 2n, are also analyzed. More sophisticated methods were developed for detecting TIM barrels specifically, including consideration of the amino acid propensities for the side chains that form the layers. We found that the residues on the outside of the eight stranded parallel beta-barrel, buried by the alpha-helices, are much more hydrophobic than the residues inside the barrel.

SUBMITTER: Nagano N

PROVIDER: S-EPMC2144152 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels.

Nagano N N Hutchinson E G EG Thornton J M JM

Protein science : a publication of the Protein Society 19991001 10

Automated methods for identifying and characterizing regular beta-barrels from coordinate data have been developed to analyze and classify various kinds of barrel structures based on geometric parameters such as the barrel strand number (n) and shear number (S). In total, we find 1,316 barrels in the January 1998 release of Protein Data Bank. Of 1,316 barrels, 1,277 barrels had an even shear number, corresponding to 50 nonhomologous families. The (beta alpha)8 triose phosphate isomerase (TIM) ba ...[more]

PMID: 10548053

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Project description:BackgroundComputational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (α/β)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts.MethodsComputational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics.ResultsExtensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (ΔG°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ΔG° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics.ConclusionsDespite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

Dataset Information

Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels.

Publications

Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels.

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