The inertia-equivalent ellipsoid: a link between atomic structure and low-resolution models of small globular proteins determined by small-angle x-ray scattering

Low-resolution three-parameter models of the shape of a biopolymer in solution can be determined by a new indirect method from small-angle X-ray scattering without contrast-variation experiments. The basic low-resolution model employed is a triaxial ellipsoid - the inertia-equivalent ellipsoid (IEE). The IEE is related to the tensor of inertia of a body and the eigenvalues and eigenvectors of this tensor can be calculated directly from the atomic coordinates and from the homogeneous solvent-excluded body of a molecule. The IEE defines a mean molecular surface (like the sea level on earth) which models the molecular shape adequately if the IEE volume is not more than 30% larger than the dry volume of the molecule. Approximately 10 to 15% of the solvent excluded volume is outside the ellipsoid; the radii of gyration of the IEE and of the homogeneous molecular body are identical. The largest diameter of the IEE is about 5 to 15% (approximately 0.2-0.8 nm) smaller than the maximum dimension of globular molecules with molecular masses smaller than 65 000 daltons. From the scattering curve of a molecule in solution the IEE can be determined by a calibration procedure. 29 proteins of known crystal structure have been used as a random sample. Systematic differences between the axes of the IEE, calculated directly from the structure, and the axes of the scattering-equivalent ellipsoids of revolution, estimated from the scattering curve of the molecule in solution, are used to derive correction factors for the axial dimensions. Distortions of model dimensions of 20 to 40% (up to 1 nm), caused by misinterpretation of scattering contributions from electron density fluctuations within the molecule, are reduced to a quarter by applying these correction factors to the axes of the scattering-equivalent ellipsoids of revolution. In a computer experiment the axes of the inertia-equivalent ellipsoids have been determined for a further nine proteins with the same accuracy. The automated estimation of the IEE from the scattering curve of a molecule in solution is realized by the Fortran77 program AUTOIEE

Engineering a mineralocorticoid to a glucocorticoid synthesizing cytochrome P450

Site-directed mutagenesis of a domain (amino acids 299-338) aligning to the I-helix region of P450cam, P450BM3 and P450terp was used to investigate the different regioselectivities displayed in the hydroxylation reactions performed by human aldosterone synthase (P450aldo) and 11beta-hydroxylase (P45011beta). The two enzymes are 93% identical and are essential for the synthesis of mineralocorticoids and glucocorticoids in the human adrenal gland. Single replacement of P450aldo residues for P45011 beta-specific residues at positions 296, 301, 302, 320, and 335 only gave rise to slightly increased 11beta-hydroxylase activities. However, a L301P/A320V double substitution increased 11beta-hydroxylase activity to 60% as compared with that of P45011 beta. Additionally substituting Ala-320 for Val-320 of P45011 beta further enhanced this activity to 85%. The aldosterone synthase activities of the mutant P450aldo proteins were suppressed to a varying degree, with triple replacement mutant L301P/E302D/A320V retaining only 10% and double replacement mutant L301P/A320V retaining only 13% of the P450aldo wild type activity. These results demonstrate a switch in regio- and stereoselectivities of the engineered P450aldo enzyme due to manipulation of residues at three critical positions, and we attribute the determination of these features in P450aldo to the structure of a region analogous to the I-helix in P450cam

Rotamers - to be or not to be? Analysis of amino acid side chain conformations in globular proteins. Side chains, regular backbone conformation, protein crystal structures

In this work the distribution of side-chain conformations in protein crystal structures is analyzed. Large deviations from rotameric #chi#-values occur systematically and cannot be attributed merely to errors in crystal structure determination. The 'rotamericity' (the fraction of residues within #+-# 20"0 of the x-angles of a rotamer) not only remains substantially below 100% (70-95% for various amino acids) with improving crystallographic resolution but actually decreases for 7 out of 17 amino acid types after a critical resolution limit is crossed. This effect has been observed for external as well as for internal residues. The rotamericity depends essentially on the different environments the amino acid meets in real protein structures. Factors such as the backbone torsion angles of the residue itself, the secondary structure and tertiary contacts influence the rotamericity. Correlations with physical properties describing volume, extension, flexibility, polarity and hydrophobicity of the side chain are shown. A substantial number of amino acid-chains are under strain. Obviously, the relaxation of some side chains is sacrified for a global minimum of free energy to be achieved at a higher organizational level involving the protein fold. The results are relevant for the development of algorithms that model proteins using restricted scanning of the torsion angle space. (WEN)Available from TIB Hannover: D.Dt.F.QN1(2,10) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman