Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions.
(9/5266)
The equilibrium unfolding of pig kidney aminoacylase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmCl, less than 1.0 M, the fluorescence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concentrations of denaturant (between 1.2 and 1.6 M GdmCl). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Further changes in the fluorescence properties occurred at higher concentrations of GdmCl, more than 1.6 M, with a decrease in emission intensity and a significant red shift of the emission maximum from 340 to 354 nm. In this stage, the secondary structure was completely broken. A study of apo-enzyme (Zn2+-free enzyme) produced similar results. However, comparison of the changes of the fluorescence emission spectra of native (Holo-) enzyme with Zn2+-free (Apo-) enzyme at low GdmCl concentrations showed that the structure of the Holo-enzyme was more stable than that of the Apo-enzyme. (+info)
Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin.
(10/5266)
Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these proteins is believed to be structural rather than enzymatic. All spectrin proteins are highly elongated and consist mainly of homologous repeats that constitute rigid segments connected in tandem. It is commonly believed that the details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this question by studying the transient electric birefringence of recombinant spectrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respectively, from Drosophila alpha-spectrin. Transient electric birefringence depends sharply on both molecular length and flexibility. We found that the birefringence relaxation time of segment 14 measured at 4 degrees C, but scaled to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rigid, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/-20%), respectively, scaled to what is expected at 20 degrees C. When the temperature is increased to 37 degrees C the main relaxation time for each of these multisegment fragments, scaled to what is expected at 20 degrees C, increased to 46, 80, and 229 ns (all +/-20%), respectively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the same as for fully extended weakly bending rods with the same lengths and axial ratios. When the ionic strength is increased to 85 mM, the main relaxation time for each of these multisegment fragments is reduced 20-50% which suggests that at physiological salt and temperature conditions the links in 2-4-segment-long fragments exhibit significant thermally induced flexing. Provided that the recombinant spectrin fragments can serve as a model for native spectrin, this implies that, at physiological conditions, the overall conformational dynamics of a native spectrin protein containing 20-40 segments equals that of a flexible polymer. (+info)
Small angle X-ray scattering of wheat seed-storage proteins: alpha-, gamma- and omega-gliadins and the high molecular weight (HMW) subunits of glutenin.
(11/5266)
Small angle X-ray scattering in solution was performed on seed-storage proteins from wheat. Three different groups of gliadins (alpha-, gamma- and omega-) and a high molecular weight (HMW) subunit of glutenin (1Bx20) were studied to determine molecular size parameters. All the gliadins could be modelled as prolate ellipsoids with extended conformations. The HMW subunit existed as a highly extended rod-like particle in solution with a length of about 69 nm and a diameter of about 6.4 nm. Specific aggregation effects were observed which may reflect mechanisms of self-assembly that contribute to the unique viscoelastic properties of wheat dough. (+info)
Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells.
(12/5266)
The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF. (+info)
Surface exposure of the methionine side chains of calmodulin in solution. A nitroxide spin label and two-dimensional NMR study.
(13/5266)
Binding of calcium to calmodulin (CaM) causes a conformational change in this ubiquitous calcium regulatory protein that allows the activation of many target proteins. Met residues make up a large portion of its hydrophobic target binding surfaces. In this work, we have studied the surface exposure of the Met residues in the apo- and calcium-bound states of CaM in solution. Complexes of calcium-CaM with synthetic peptides derived from the CaM-binding domains of myosin light chain kinase, constitutive nitric-oxide synthase, and CaM-dependent protein kinase I were also studied. The surface exposure was measured by NMR by studying the effects of the soluble nitroxide spin label, 4-hydroxyl-2,2,6, 6-tetramethylpiperidinyl-1-oxy, on the line widths and relaxation rates of the Met methyl resonances in samples of biosynthetically 13C-methyl-Met-labeled CaM. The Met residues move from an almost completely buried state in apo-CaM to an essentially fully exposed state in Ca2+4-CaM. Binding of two Ca2+ to the C-terminal lobe of CaM causes full exposure of the C-terminal Met residues and a partial exposure of the N-terminal Met side chains. Binding of the three target peptides blocks the access of the nitroxide surface probe to nearly all Met residues, although the mode of binding is distinct for the three peptides studied. These data show that calcium binding to CaM controls the surface exposure of the Met residues, thereby providing the switch for target protein binding. (+info)
Automated MAD and MIR structure solution.
(14/5266)
Obtaining an electron-density map from X-ray diffraction data can be difficult and time-consuming even after the data have been collected, largely because MIR and MAD structure determinations currently require many subjective evaluations of the qualities of trial heavy-atom partial structures before a correct heavy-atom solution is obtained. A set of criteria for evaluating the quality of heavy-atom partial solutions in macromolecular crystallography have been developed. These have allowed the conversion of the crystal structure-solution process into an optimization problem and have allowed its automation. The SOLVE software has been used to solve MAD data sets with as many as 52 selenium sites in the asymmetric unit. The automated structure-solution process developed is a major step towards the fully automated structure-determination, model-building and refinement procedure which is needed for genomic scale structure determinations. (+info)
Discrimination of solvent from protein regions in native Fouriers as a means of evaluating heavy-atom solutions in the MIR and MAD methods.
(15/5266)
An automated examination of the native Fourier is tested as a means of evaluation of a heavy-atom solution in MAD and MIR methods for macromolecular crystallography. It is found that the presence of distinct regions of high and low density variation in electron-density maps is a good indicator of the correctness of a heavy-atom solution in the MIR and MAD methods. The method can be used to evaluate heavy-atom solutions during MAD and MIR structure solutions and to determine the handedness of the structure if anomalous data have been measured. (+info)
Solution structure of BID, an intracellular amplifier of apoptotic signaling.
(16/5266)
We report the solution structure of BID, an intracellular cross-talk agent that can amplify FAS/TNF apoptotic signal through the mitochondria death pathway after Caspase 8 cleavage. BID contains eight alpha helices where two central hydrophobic helices are surrounded by six amphipathic ones. The fold resembles poreforming bacterial toxins and shows similarity to BCL-XL although sequence homology to BCL-XL is limited to the 16-residue BH3 domain. Furthermore, we modeled a complex of BCL-XL and BID by aligning the BID and BAK BH3 motifs in the known BCL-XL-BAK BH3 complex. Additionally, we show that the overall structure of BID is preserved after cleavage by Caspase 8. We propose that BID has both BH3 domain-dependent and -independent modes of action in inducing mitochondrial damage. (+info)