Ordered macromolecular structures in ferrofluid mixtures.
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Journal Article↗
Biomedical subjects
J Trewhella
Publications and source records attributed to J Trewhella.
Glycation of calmodulin: chemistry and structural and functional consequences.
In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylyl Cyclases↗
Calmodulin and troponin C structures studied by Fourier transform infrared spectroscopy: effects of Ca2+ and Mg2+ binding.
Fourier transform infrared (FTIR) spectroscopy has been used to examine the conformationally sensitive amide I' bands of calmodulin and troponin C. These are observed to undergo a sequence of spectroscopic changes which reflect conformational rearrangements that take place when Ca2+ is bound. Calmodulin and troponin C show similar though not identical changes on Ca2+ binding, and the effect of Mg2+ on troponin C is quite different from that of Ca2+. Both proteins show absorption maxima in the amide I' region at 1644 cm-1 which is significantly lower in frequency than has been generally observed for proteins that contain a high percentage of alpha-helix. It is proposed that an unusually high proportion of the helices in the structures of these proteins are distorted from the normal alpha-helical configuration such that the carbonyl stretching frequencies are lowered. It is further proposed that the shift to lower frequency is due to backbone carbonyl groups in the distorted helices that form strong hydrogen bonds with solvent molecules. A decrease in intensity at 1654 cm-1, the normal frequency assignment for alpha-helical structure, is observed as Ca2+ binds to calmodulin and troponin C. This suggests that Ca2+ binding results in a net decrease in "normal" alpha-helix conformation. There is a corresponding increase in intensity of the band at 1644 cm-1, possibly due to an increase in distorted helix content, allowing for a net increase in helix consistent with circular dichroism estimates of the Ca2+-dependent changes in helix content in calmodulin.
Animals↗
Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering.
While X-ray crystallographic data on cytochrome c show the reduced and oxidized forms to have very similar structures, there is a considerable body of data, mostly from solution studies, that indicates the reduced form is more stable and that the interior of the protein is less accessible to solvent in this state. These observations have led to the hypothesis that while the time-averaged structure is preserved between the two forms, the dynamics of the two forms are different. The oxidized form has been proposed to undergo more large-amplitude, low-frequency motions than the reduced form. The crystal structure data were derived from crystals grown in high salt concentrations, but the solution studies were done at relatively low ionic strength. Small-angle X-ray scattering has been used to examine the effects of the ionic strength and oxidation state on the solution structure of cytochrome c. We find that the radius of gyration and the maximum linear dimension of oxidized cytochrome c are significantly larger than those for reduced cytochrome c, in 5 mM phosphate buffer at pH 7.3, and further that this difference is suppressed by addition of 200 mM sodium chloride. We conclude that there is a real structural difference between the two forms at low ionic strength in solution and that this difference is likely to contribute to the observed differences in accessibility and compressibility.
Cytochrome c Group↗
Comparison of the crystal and solution structures of calmodulin and troponin C.
X-ray solution scattering data from skeletal muscle troponin C and from calmodulin have been measured. Modeling studies based on the crystal structure coordinates for these proteins show discrepancies between the solution data and the crystal structure that indicate that if the size and shape of the globular domains are the same in solution as in the crystal, the distances between them must be smaller by several angstroms. Bringing the globular domains closer together requires structural changes in the interconnecting helix that joins them.
Animals↗
Characterization of metal binding peptides from cadmium resistant plant cells.
The majority of the cellular cadmium (less than 80%) in cadmium resistant Datura innoxia cells is bound to a small, metal induced peptide which is not metallothionein. This peptide consists of glutamate, cysteine and glycine in a ratio between 2:2:1 and 3:3:1 and has an apparent molecular weight of 776, under denaturing conditions. It is heat stable and complexes with cadmium to produce multimeric forms which are separable by gel filtration. Chemical analyses suggest that some amino acids are not joined by classical peptide linkages. This indicates that the synthesis of the peptide may not be directed by mRNA and that induction of its synthesis may not involve increased transcription from a putative gene corresponding directly to this peptide. A smaller proportion (greater than 15%) of the cellular cadmium is bound to a larger compound which is also heat stable and binds copper more readily than cadmium in vivo. This larger compound has an amino acid composition similar, in some respects, to metallothioneins.
Amino Acids↗
Reformation of crystalline purple membrane from purified bacteriorhodopsin fragments.
Reconstituted crystalline purple membrane has been prepared starting from denatured bacteriorhodopsin (BR) fragments, native lipids and retinal. The two chymotryptic fragments are thought to contain respectively five and two transmembrane alpha-helices in native BR. The new reconstitution procedure, a modification of that of Huang et al. (1986, J. Biol. Chem., 256, 3802), relies on dodecylsulfate precipitation by potassium ions and yields samples with a high protein-to-lipid ratio (approximately 1:1 w/w). X-ray and neutron diffraction measurements show that in the reconstituted samples BR molecules are arranged in a P3 two-dimensional lattice with the same unit cell dimensions as the native purple membrane lattice. Analysis of reflection intensities indicates that the reconstituted molecules have regained the structure of native BR to 7 A resolution.
Bacteriorhodopsins↗
Localization of two chymotryptic fragments in the structure of renatured bacteriorhodopsin by neutron diffraction.
The structure of crystalline purple membrane reconstituted from purified bacteriorhodopsin (BR) chymotryptic fragments has been studied by neutron diffraction. In one of the samples studied, the fragment C-2, encompassing the first two predicted transmembrane segments, was prepared from deuterated purple membrane. The diffraction changes when the natural C-2 fragment is substituted by a deuterated one are analysed in terms of a seven-helix model for BR. The assignment of the labelled fragment to one end of the molecule placed new constraints on folding models for the protein.
Bacteriorhodopsins↗
Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis.
Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated 3H leucine from the digested membranes. During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.
Amino Acid Sequence↗
Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map.
Specific amino acid sequence segments have been assigned to locations in the structural map of bacteriorhodopsin using two-dimensional neutron diffraction data and a model building analysis. Models are constructed computationally by building specific regions of the amino acid sequence as alpha helices and then positioning the helices on axes indicated by the density map of Henderson and Unwin (Nature [Lond.]. 1975, 257:28-32). Neutron diffraction data were collected from samples of stacked, oriented "native" purple membranes as well as purple membranes containing different kinds of deuterated amino acids. Models differing in the assignments of helices to specific axes and in rotations of the helices about those axes were tested against the neutron data using a weighted residual factor to rank the models. This residual factor was calculated between observed and predicted intensity differences for pairs of data sets. Using this approach, a small set of related models has been found that predicts the observed intensity changes between five independent data sets. These models are inconsistent with the proposed locations of the retinal chromophore and the carboxyl terminus and with any of the previously proposed models for bacteriorhodopsin.
Amino Acid Sequence↗
1H-NMR studies of ferric soybean leghemoglobin: assignment of hyperfine shifted resonances of complexes with cyanide, nicotinate, pyridine and azide.
The 1H-NMR spectra of complexes of soybean ferric leghemoglobin a with cyanide, nicotinate, pyridine and azide have been recorded. Assignments of many of the hyperfine shifted resonances to specific groups on the periphery of the heme have been made on the basis of their intensities and chemical shifts, pH dependence, nuclear Overhauser effects, spin decoupling and the use of Gd3+ as a relaxation probe. The resonances of the protons at positions 3 and 5 of pyridine and nicotinate ligands have also been assigned. The iron(III) atom in the cyanide, nicotinate and azide complexes is almost completely low spin. In the pyridine complex, which is predominantly low spin, a high-spin state is thermally populated at room temperature. Information on the conformation of the heme propionate and vinyl side-chains is obtained. The average rotational position of one of the heme vinyl groups appears to differ between the nicotinate and cyanide complexes. In both of these complexes conformational rearrangement of a heme propionic acid side-chain occurs upon deprotonation of its carboxylic acid group (pK approx. 5.0). A further change in the conformation of this group occurs in leghemoglobin nicotinate upon deprotonation of the distal histidine. The pK of the heme propionic acid side-chain in leghemoglobin pyridine is 5.6. Its conformation and environment appears to differ from that in the nicotinate and cyanide complexes. In leghemoglobin cyanide, evidence for an interaction between the protonated distal histidine and the cyanide ligand is obtained. In each of the complexes studied the unpaired electron spin distribution over the porphyrin ring is highly anisotropic. Considerable differences exist between the unpaired spin distribution in the cyanide complexes of leghemoglobin and myoglobin. The axial ligand field is stronger in leghemoglobin and this may be of significance in the reaction with oxygen.
Azides↗
Spin state equilibria in soybean ferric leghemoglobin and its complexes with fomate and acetate.
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Acetates↗
The solution structures of calmodulin and its complexes with synthetic peptides based on target enzyme binding domains.
Small-angle X-ray and neutron scattering experiments have given important information on the solution structures of calmodulin and its complexes with synthetic peptides used to model target enzyme interactions. In combination with crystallographic data, site directed mutagenesis and various spectroscopic studies, these experiments have contributed to our understanding of the solution structure of calmodulin in different functional states. We have gained important insights into the conformational flexibility in calmodulin that appears to be crucial to its regulatory functions. Specifically, flexibility in the interconnecting helix region of calmodulin has been shown to play a critical role in facilitating calmodulin's binding to a wide variety of target enzymes whose activities are thus regulated. This review will focus mainly on the contributions small-angle scattering has made to our understanding of the solution structure of calmodulin in the context of other studies, with particular regard to circular dichroism and Fourier transform infrared studies that complement the small-angle scattering data.
Binding Sites↗