Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases

The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments

We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)].     

Formation of giant nuclei in continuously irradiated tumors

Summary Transplantable malignant tumors of mice and rats have been irradiated continuously for several days by means of embedded glass beads containing Sr⁹⁰-Y⁹⁰. Concomitant enlargement of tumor cell nuclei occurs in areas where mitosis is inhibited, i.e. associated with doses of 1200–2000 rads per day and above. Some enlarged cells survive at least a week in areas where the dose rate is much higher than this. Intense incorporation of thymidine into enlarged cells can be seen at least a week after beginning irradiation.Work supported by the U.S. Atomic Energy Commission.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his eightieth birthday.     

AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long‐lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.     

Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction.

Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin. Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment. This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium. Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5. Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH. D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH. R82Q showed diminished proton pumping with the same pH dependence as for wild type. Bacteriorhodopsin purified from E. coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants. One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches. The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg.