Amino acid residues 4425-4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor.

We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor.

Three-dimensional location of the imperatoxin A binding site on the ryanodine receptor.

Cryo-electron microscopy and three-dimensional, single-particle image analysis have been used to reveal the specific binding site of imperatoxin A (IpTx(a)) on the architecture of the calcium release channel/ryanodine receptor from skeletal muscle (RyR1). IpTx(a) is a peptide toxin that binds with high affinity to RyR1 and affects its functioning. The toxin was derivatized with biotin to enhance its detection with streptavidin. IpTx(a) binds to the cytoplasmic moiety of RyR1 between the clamp and handle domains, 11 nm away from the transmembrane pore. The proposed mimicry by IpTx(a) of the dihydropyridine receptor (DHPR) II-III loop, thought to be a main physiological excitation-contraction trigger, suggests that the IpTx(a) binding location is a potential excitation-contraction signal transduction site.

Contributions of electron microscopy and single-particle techniques to the determination of the ryanodine receptor three-dimensional structure.

The ryanodine receptor is the main intracellular calcium release channel from the sarcoplasmic reticulum in striated muscle. It is the largest ion channel known, composed of four identical major subunits of 565 kDa and four smaller 12-kDa subunits, identified as FK-506 binding protein. The successful isolation of the ryanodine receptor together with the development of cryoelectron microscopy and single-particle image processing techniques have enabled major progress to be made in the determination of the receptor's structure over the past decade. Three-dimensional reconstruction shows the receptor to be composed of two main parts, a large square shaped cytoplasmic assembly and a smaller transmembrane assembly. The cytoplasmic assembly has an unusual architecture in which about 10 domain-like structures are interconnected in a loosely packed manner. Subsequent studies have started to reveal conformational changes associated with channel gating and the localization of binding sites for some proteins with which the receptor interacts (calmodulin, and FK-506 binding protein). It is becoming clear that long-range induced conformational changes must be involved in the mechanisms of modulation of the receptor's gating properties.

Structural characterization of a dynein motor domain.

Cytoplasmic dynein is a microtubule-based mechanochemical protein that plays an essential role in cell division, vesicle transport, and cytoplasmic membrane organization. As a molecular motor, dynein utilizes an ATP hydrolysis mechanism to bind and release microtubules and to undergo conformational changes that result in a net displacement towards the microtubule's minus end. To visualize structural features of this motor protein, we have begun to characterize the dynein head domain by electron microscopy and image processing. Transmission electron microscopy of negatively stained native dynein from Dictyostelium has been performed and images of the head domain have been aligned and analyzed with the software SPIDER. The resulting 2D averages show an oblong round shape composed of seven to eight globular domains or lobes that encircle a stain-filled area. A recombinant 380 kDa fragment of the dynein heavy chain encodes just the globular head domain; analysis of these particles reveals a high structural similarity with the native head domain. A prominent stalk can be seen in several projections of this fragment, suggesting a structure analogous to the B-link described for some axonemal dyneins. Single tilt pair images were used to compute low resolution 3D reconstructions of the dynein head domain. These show a flattened spheroidal shape of 13.5 nm in length with seven similar domains arranged in a ring. Slices through the reconstructions reveal a large central cavity. This is the first detailed description of the head domain structure for a dynein molecule. The presence of a central cavity and the outer globular features, along with its large size make dynein structurally distinct from either myosin or kinesin.

Overexpression of cytoplasmic dynein's globular head causes a collapse of the interphase microtubule network in Dictyostelium.

Cytoplasmic dynein is a minus-end directed microtubule-based motor. Using a molecular genetic approach, we have begun to dissect structure-function relationships of dynein in the cellular slime mold Dictyostelium. Expression of a carboxy-terminal 380-kDa fragment of the heavy chain produces a protein that approximates the size and shape of the globular, mechanochemical head of dynein. This polypeptide cosediments with microtubules in an ATP-sensitive fashion and undergoes a UV-vanadate cleavage reaction. The deleted amino-terminal region appears to participate in dimerization of the native protein and in binding the intermediate and light chains. Overexpression of the 380-kDa carboxy-terminal construct in Dictyostelium produces a distinct phenotype in which the interphase radial microtubule array appears collapsed. In many cells, the microtubules form loose bundles that are whorled around the nucleus. Similar expression of a central 107-kDa fragment of the heavy chain does not produce this result. The data presented here suggest that dynein may participate in maintaining the spatial pattern of the interphase microtubule network.

Evidence for sodium dodecyl sulfate/protein complexes adopting a necklace structure.

Structural analysis by cryo-electron microscopy and small-angle X-ray scattering of ten sodium dodecyl sulfate/protein complexes in 25 mM Tris/HCl, 0.192 M glycine, pH 8.3, showed necklace-like structures of spherical micelles dispersed along the unfolded peptide chain. The micelles of most SDS/protein complexes had a constant diameter (approximately 6.2 nm), slightly larger than pure SDS micelles (approximately 5.7 nm), all micelles possessing a degree of surface roughness. The micelle-associated polypeptide is mostly situated at the interface of the sulfate head groups and hydrocarbon core, intruding into the core rather than outward from the surface. Proteins with a molecular mass less than about 20 kDa formed complexes with a single SDS micelle. Multi-micellar SDS/protein complexes had centre-to-centre intermicellar distances in the range 7.0-12.0 nm. Our findings on the constancy of micellar size, number of micelles/complex, and the relationship between the degree of occupancy of micelles and a polypeptide's molecular mass, have enabled us to speculate on the correlation between the electrophoretic mobility of a polypeptide in SDS/PAGE and its molecular mass. The anomalous electrophoretic behaviour observed for the sodium dodecyl sulfate/histone H5 complex is accounted for by the large micelle of its complex.

Unfolded structure and reactivity of nucleosome core DNA-histone H2A,H2B complexes in solution as studied by synchrotron radiation X-ray scattering.

It has been previously found using different physicochemical techniques [Aragay, A., Diaz, P., & Daban, J.-R. (1988) J. Mol. Biol. 204, 141-154] that histones H2A,H2B in the absence of H3,H4 can associate with nucleosome core DNA (146 base pairs). Here we describe a synchrotron X-ray scattering study of core DNA-(H2A,H2B) complexes in solution. Our results obtained using different histone to DNA weight ratios and ionic conditions ranging from very low ionic strength to 0.2 M NaCl show that histones H2A,H2B are unable to fold core DNA. Model calculations indicate that histones H2A,H2B produce very elongated structures even when the reconstituted complexes are prepared at physiological ionic strength. In contrast, our scattering data indicate that the reconstituted complexes prepared at physiological salt concentration either with the four core histones or with histones H3,H4 without H2A,H2B are completely folded particles with a radius of gyration similar to that corresponding to the native nucleosome core (4.2 nm). Furthermore, our results show that the DNA of the extended complexes containing histones H2A,H2B becomes completely folded after the histone pair exchange reaction that occurs spontaneously between preformed DNA-(H2A,H2B) and DNA-(H3,H4) complexes. These observations, together with our previous studies, suggest that the open conformation of DNA-(H2A,H2B) complexes facilitates the involvement of this structure as a transient intermediate in the reaction of nucleosome formation at physiological ionic strength.

Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels.

In a previous work (J.-R. Daban, M. Samsó, and S. Bartolomé, Anal. Biochem. 199, 162-168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[alpha]-phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 M Tris and 0.192 M glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680-685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein-SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethyl-rhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass.

Use of nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.