Relationship between intelligence and the size and composition of the corpus callosum.

We investigated the relationship between the morphology of the corpus callosum (CC) and IQ in a healthy sample of individuals in their late teens and early twenties. The relationship between the area of the CC, measured at the midline, and IQ showed regional differences. We observed that a higher estimated performance IQ was associated with smaller area in the posterior regions of the CC, a finding that differs from a positive association previously observed in a somewhat older adult sample. In contrast, higher estimated verbal IQ was associated with decreased fractional anisotropy of the genu, an anterior portion of the CC. Age effects were also observed such that older age was associated with larger CC area. Our results suggest that CC morphology is related to cognitive performance, which may have implications for clinical populations in whom CC morphology is atypical.

Attentional selection and the processing of task-irrelevant information: insights from fMRI examinations of the Stroop task.

In this chapter, we discuss our research that reveals how attentional mechanisms can modulate activity of posterior brain regions responsible for processing the unattended attribute of a stimulus. To do so, we utilized fMRI to reveal patterns of regional brain activity for variants of the Stroop task that differ in the nature of the task-irrelevant stimulus attribute. In all variants, individuals had to identify the ink color in which an item was presented. To vary attentional demands, we manipulated whether or not the task-irrelevant information contained conflicting color information. The variants differed in whether the conflicting color information was contained in a word naming a color (e.g. the word 'red' in blue ink), a word naming an object highly associated with a specific color (e.g. the word 'frog' in red ink), or a line drawing of an object highly associated with a specific color (e.g. a drawing of a frog in red ink). When the unattended stimulus attribute contained color information that conflicted with an item's ink color, increased activity was observed in the posterior brain region that processes the aspect of the task-irrelevant attribute related to color. Increased activity was observed in the left precuneus and left superior parietal cortex when the conflicting information arose from a color word; in the middle temporal gyrus and insular cortex when the word named an object highly associated with a specific color, and included extensive regions of early portions of the ventral visual processing stream when a line drawing was highly associated with a specific color. These areas have been implicated in word processing, semantic processing, and visual processing, respectively. Our results suggest that attentional selection can occur by: (1) increasing the gain on all posterior regions responsible for processing information related to the task demands, regardless of whether that information is contained in the task-relevant or task-irrelevant dimension; (2) limiting the processing of task-irrelevant information in order to reduce interference; and (3) modulating the processing of representations varying from those of a low-level perceptual nature up through those of a higher-order semantic nature.

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

Crystallization and preliminary crystallographic analysis of E. coli uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase in two new crystal forms.

Uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase (MurB), the second enzyme in the peptidoglycan synthetic pathway of Escherichia coli, has been crystallized in two previously unreported forms, one orthorhombic and the other monoclinic. MurB (molecular mass 38 kDa) crystallizes in a range of conditions that utilize polyethylene glycol fractions as precipitants, and crystals can be grown with or without the enzyme's substrate, uridine 5'-diphospho-N-acetylenolpyruvylglucosamine. X-ray diffraction from crystals of the orthorhombic form extends to 2 A resolution and shows the symmetry and systematic absences of space group P2(1)2(1)2(1). These crystals show significant variations in cell dimensions at room temperature and at 100 K. A crystal used to collect a 2.0 A resolution data set at a synchrotron source showed cell dimensions at ca 100 K of a = 51.0, b = 79.3 and c = 87.1 A, indicating one molecule peroasymmetric unit. The monoclinic crystals scatter X-rays to 3.0 A resolution consistent with space group P2(1), unit-cell dimensions (ca 100 K) a = 50.7, b = 92.4, c = 85.5 A, and beta = 104 degrees, and two molecules per asymmetric unit. Mercury derivatives have been prepared with both orthorhombic and monoclinic forms, and efforts are underway to exploit these derivatives to determine the structure of this protein.

Three-dimensional structure of the muscle fatty-acid-binding protein isolated from the desert locust Schistocerca gregaria.

The three-dimensional structure of the fatty-acid-binding protein isolated from the flight muscle of the desert locust Schistocerca gregaria has been solved and refined to a crystallographic R-value of 18.5% for all measured X-ray data from 30.0- to 2.2-A resolution. Crystals employed in the investigation were grown from 2.6 to 2.8 M ammonium sulfate solutions, buffered at pH 7.5 and containing 2-5% 2-methyl-2,4-pentanediol. They belonged to the space group P2(1) with unit cell dimensions of a = 61.6 A, b = 44.8 A, c = 63.9 A, and beta = 113.6 degrees and two molecules per asymmetric unit. The protein fold consists of ten strands of antiparallel beta-pleated sheet that wrap around to form a beta-barrel. In addition, there are two small alpha-helices and six type I, two type II, and two type II' turns. The two molecules pack in the asymmetric unit as a dimer with a local 2-fold rotational axis. The subunit-subunit interface involves amino acid side chains located in the area of the helix-turn-helix motif and the turn between beta-strands E and F. It is this area that has been speculated to form the portal through which fatty acids enter the binding cavity. There are 23 solvent molecules that are conserved between the two independent molecules in the asymmetric unit. Nine of these waters play important structural roles. A three-dimensional comparison between the insect and human muscle fatty-acid-binding proteins shows that their alpha-carbons superimpose with a root-mean-square deviation of 0.77 A for 89 structurally equivalent atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function studies of [2Fe-2S] ferredoxins.

The ability to overexpress [2Fe-2S] ferredoxins in Escherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegatative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in in these proteins in their oxidized and reduced states has been studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

Molecular structure of the oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120 determined to 1.7-A resolution.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. The three-dimensional structure of this ferredoxin has now been determined and refined to a crystallographic R value of 16.7%, with all measured X-ray data from 30.0 to 1.7 A. The molecular motif of this ferredoxin is similar to that of other plant-type ferredoxins with the iron-sulfur cluster located toward the outer edge of the molecule and the irons tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteinyl residues. The overall secondary structure of the molecule consists of seven strands of beta-pleated sheet, two alpha-helices, and seven type I turns. It is of special interest that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different in the heterocyst form of the protein. Three of these positions are located in the metal-cluster binding loop.

Crystallization and preliminary analysis of oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.

Interdomain salt bridges modulate ligand-induced domain motion of the sulfate receptor protein for active transport.

The refined crystal structure of the liganded form of the Salmonella typhimurium sulfate-binding protein, a periplasmic receptor of active transport, is made up of two globular domains bisected by a deep cleft wherein the dehydrated sulfate is completely engulfed and bound by hydrogen bonds and van der Waals' forces. Two salt bridges (between Glu15 and Arg174 and between Asp68 and Arg134) span the cleft opening. To elucidate the role of the inter-domain salt bridges in the ligand-induced domain motion, the acidic residues were changed (singly and together) to their corresponding amide side-chains by site-directed mutagenesis of the recombinant Escherichia coli sulfate-binding protein. Rapid kinetics and equilibrium measurements of sulfate binding to the purified mutant proteins demonstrate that these salt bridges stabilize the closed liganded form of the receptor and modulate the rate of cleft opening. Our results have new implications in understanding the dynamics of many other multidomain proteins that undergo similar large-scale domain motions.

Two-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin. Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the 2Fe-2S* cluster.

Hyperfine 1H NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W.R., Breiter, D.R., Benning, M.M., Wesenberg, G., Oh, B.-H., Markley, J.L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 1H alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies.

Using recombinant DNA techniques, an Escherichia coli periplasmic sulfate receptor or sulfate-binding protein involved in active transport has been overexpressed and characterized. This protein is essentially identical in size, sequence, antigenicity, and ligand affinity and specificity to the sulfate receptor from Salmonella typhimurium whose crystal structure has been refined at 2 A resolution. The dehydrated sulfate is bound in the deep cleft between the two lobes of the bilobate protein. Using the structure of the S. typhimurium as a guide, three site-directed mutants (Ser129Cys, Gly46Cys, and Ser129Cys/Gly46Cys) have been made. In the Cys129/Cys46 mutant the disulfide has been successfully introduced across the opening of the ligand-binding site cleft of the E. coli sulfate-binding protein. The dissociation of sulfate from the double mutant protein is very slow under oxidizing conditions and increases more than 200-fold when reducing agent is added. This effect is attributed to a loss of interdomain structural flexibility in the presence of the disulfide, and underscores the importance of protein conformational change in binding protein function.

The calcium-binding site in the galactose chemoreceptor protein. Crystallographic and metal-binding studies.

We have determined the relative affinities in solution for various metals which bind to the lone calcium-binding site of the D-galactose-binding protein which resembles the EF-hand loop. In order of affinity the metals are: Ca2+ approximately Tb3+ approximately Pb2+ greater than Cd2+ greater than Sr2+ greater than Mg2+ much greater than Ba2+. The binding affinity for calcium (Kd = 2 microM) and the slow off-rate determined for terbium (1 x 10(-3) s-1) and that the metal-binding site is unperturbed by sugar binding argue for a structural role. Furthermore, we have crystallographically refined the structure of the binding protein with the calcium substituted by cadmium, compared it with the calcium-bound structure, and found them to be identical. The results of these structural and solution studies support the hypothesis that for a given metal-binding loop, cation hydration energy, size, and charge are major factors contributing to binding affinity.

Sulfate-binding protein dislikes protonated oxyacids. A molecular explanation.

We have determined the effect of pH on the binding affinities of the conjugate bases of four different tetrahedral oxyacids to the sulfate-binding protein. The equilibrium dissociation constants of the binding of sulfate (Kd = 0.12 microM) and selenate (Kd = 5 microM) were found to be pH independent over the range pH 5 to pH 8.1, whereas chromate binding exhibited a pH dependence that is approximately attributable to the pK2 of the chromic acid. Phosphate was bound with an affinity five orders of magnitude weaker than that of sulfate. In light of the highly refined 2 A structure of the complex of the sulfate-binding protein with sulfate, and considering the protonation state and net charge of the various oxyacids, we conclude that the pH dependence of chromate binding and the extremely low affinity of phosphate are attributable mainly to a lack of hydrogen bond acceptors in the binding site. These studies demonstrate that the sulfate-binding site is stringently designed to bind tightly tetrahedral, fully ionized, oxyacid dianions. The presence of a donatable proton on the ligand reduces binding energy by approximately 7 kcal/mol.