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R M Garavito

Publications and source records attributed to R M Garavito.

At least 55 records · Page 3Linked to original sources
PubMed1993

Imaging of reconstituted biological channels at molecular resolution by atomic force microscopy.

Using atomic force microscopy (AFM), we obtained high-resolution surface images of the bacterial outer membrane channels Escherichia coli OmpF porin and Bordetella pertussis porin that were reconstituted in artificial bilayer membranes as two-dimensional crystalline arrays. These porins were chosen because they are among the most extensively studied proteins of this type and are known for their well-defined crystalline nature in the native membrane. Such reconstituted membrane proteins are ideal specimens to assess the suitability and resolution of AFM for imaging biomembranes and associated proteins. Although OmpF porin often showed a mixed pattern of rectangular and hexagonal arrays with approximately 8.4 x 9.8- and approximately 7.2-nm-spacings, respectively, B. pertussis porin showed mostly a rectangular pattern with an approximately 7.9 x 13.8-nm spacing. The packing patterns of the E. coli OmpF porin in the membrane are very close to those found in electron-microscopic studies. When B. pertussis porin was imaged in a buffer solution, its trimeric subunits were apparently resolved, and the surface of each monomer revealed beadlike structures. This is the first report of such a high-resolution structural analysis of B. pertussis porin by any imaging method. We also imaged the lipid bilayer itself as an internal control for imaging and to further ascertain the resolution. Individual polar head groups of bilayer lipid molecules were resolved, suggesting the intrinsic resolution of AFM for bioimaging.

Bordetella pertussis↗
PubMed1988

The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation.

The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane.

Bacterial Outer Membrane Proteins↗
PubMed1987

Crystallization of the photosystem I reaction centre.

The reaction centre of the photosynthetic membrane complex photosystem I (PSI) from the thermophilic cyanobacterium Phormidium laminosum was found to crystallize under a range of conditions. The crystallization method, which can occur in the presence of larger detergent molecules than those used previously for the crystallization of membrane proteins, is presented in this report. Several crystal forms have been observed, and some of these show birefringence and linear dichroism. Optical measurements on crystals thicker than 5 microm were severely restricted because of the very high chlorophyll density within the crystals, but linear dichroism measurements on thin single crystals were possible and the results are presented here. By comparing the data with earlier measurements on oriented PSI complexes, a working model for the orientation of the PSI complexes within the crystal could be proposed. The PSI reaction centre is one of the largest and most complex membrane protein units that have been crystallized to date.

Journal Article↗
PubMed1986

Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions.

Thermal and low pH stabilities of matrix porin (Omp F) solubilized in the micellar solutions of ionic (SDS) and nonionic detergents were investigated by the methods of circular dichroism, intrinsic fluorescence, light scattering and sedimentation velocity. The stability of porin structure in solution is much higher in the presence of beta-octyl glucoside than with SDS. In the presence of SDS, sharp transitions were detected by all parameters measured, above 55 degrees C at neutral pH and below pH 4.5 at 20 degrees C. These transitions involve at least three concomitant processes: unfolding of protein, dissociation of trimers to monomers and the disruption of the protein-detergent micellar complexes, all events being irreversible in the presence of SDS. The nonionic detergent, beta-octyl glucoside, increases the stability of porin in acidic conditions, since neither dissociation nor denaturation was observed in the pH region between 7.5 and 2.0. However, at pH less than 3.5, small, reversible changes in protein structure became evident. The thermal stability of porin is also increased by beta-octyl glucoside as evidenced by a transition temperature 15-20 degrees C higher as compared to SDS. A considerable degree of native porin structure was regained after heat treatment in the presence of beta-octyl glucoside, though the reconstituted trimers were not identical to the native ones. The addition of lipopolysaccharide and divalent cations (Ca2+, Mg2+) to the experimental system did not improve the thermal reversibility.

Bacterial Outer Membrane Proteins↗
PubMed1985

Secondary structure of a channel-forming protein: porin from E. coli outer membranes.

Porin from Escherichia coli outer membranes has been analysed by high angle diffuse X-ray diffraction, and by attenuated total reflection infrared spectroscopy. These methods demonstrate independently that the majority of the polypeptide backbone is arranged in anti-parallel beta-pleated sheet structure. The average length of the beta-strands, which are oriented nearly normal to the membrane plane, is estimated to be 10-12 residues, independent of the method used. Although the details of strand arrangement (beta-barrels or stacked sheets) are not as yet known, porin represents the first transmembrane protein for which beta-structure has been established unequivocally.

Bacterial Outer Membrane Proteins↗
PubMed1984

The crystallization of outer membrane proteins from Escherichia coli. Studies on lamB and ompA gene products.

The outer membrane protein LambB from Escherichia coli has been crystallized from detergent-containing solutions. Several different crystal habits can be obtained under the same ionic and precipitant conditions by altering the detergent head group composition of the protein-detergent mixed micelle or by adding polar organic compounds. Two crystal forms have been partially characterized as P1 and C2221, the former diffracting to beyond 4 A resolution and the latter to 6 A. The detergents used were beta-octyl glucoside, octyl tetraoxyethylene, and octyl polyoxyethylene (polydisperse) either alone or as mixtures. In some experiments, the addition of small nonionic amphiphiles having n-butyl alkyl tails significantly influenced crystallization. The experiments suggest that the detergent region of the mixed micelle plays a critical role in crystal formation. Using the methods developed here for LamB and also for matrix porin (Garavito, R. M., Jenkins, J. A., Jansonius, J. N., Karlsson, R., and Rosenbusch, J. P. (1983) J. Mol. Biol. 164, 313-327), an additional protein from the outer membrane, OmpA, has been obtained as a microcrystalline preparation.

Bacterial Outer Membrane Proteins↗
PubMed1983

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes.

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.

Bacterial Proteins↗
PubMed1983

Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique.

Qualitative and quantitative tests were performed to determine whether the temperature at which dehydration and embedding occur affects the antigenic specificity of tubulin and the protein A-gold (pAg) immunolabeling technique. The analysis indicates that low temperature (-35 degrees C) treatment increased the specificity and density of pAg labeled anti-tubulin antibodies to Leishmania tropica subpellicular microtubules as compared to samples prepared at 0 degrees C or 20 degrees C.

Acrylic Resins↗
PubMed1982

Biochemical and structural analyses of microtubules in the pellicular membrane of Leishmania tropica.

The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the alpha and beta subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysis by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the beta subunits of Leishmania and pig tubulin have very similar primary structures, while the alpha subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are alpha and beta subunits of tubulin. Immunoelectron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12 degrees. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.

Amino Acid Sequence↗
PubMed1982

Perspectives for achieving improved information by the observation of thin sections in the electron microscope.

Obtaining biologically significant fine detailed information from sections is limited firstly by the unknown distribution of heavy metal stain and secondly by conformational changes induced by dehydration. Only afterwards we have to be concerned with beam induced alterations. By Z-imaging in a STEM, completely unstained material can now become imaged sharply with high contrast. By this the first limitation is eliminated and the second can become explored. For this purpose new resins designed for low temperature embedding might become important. First biological results are presented which illustrate the potential of these techniques: The transmembrane protein of the separate junction has been revealed as such and shown that only the hydrophilic part of the protein is stained by uranyl acetate. This type of staining therefore does not allow the detection of any transmembrane proteins in sections.

Animals↗
PubMed1982

Structural investigations of outer membrane proteins from Escherichia coli.

The receptor of phage lambda of Escherichia coli W3110, and matrix porin from E. coli BE have been crystallized. Porin occurs in two crystal forms whose packing arrangements are different, allowing the conclusion that crystal growth occurs from monodisperse protein-detergent complexes. The tetragonal form yields resolution to 2.9 A. The hexagonal form allows conclusive support for the hypothesis that a large fraction of the polypeptide is present in beta-pleated sheet structure with the strands nearly parallel to the normal of the membrane plane.

Bacterial Proteins↗
PubMed1980

Three-dimensional crystals of an integral membrane protein: an initial x-ray analysis.

Matrix protein, a pore-forming transmembrane protein spanning the outer membrane of Escherichia coli, has been obtained in a variety of three-dimensional crystal forms amenable to both electron microscope and x-ray analyses. Successful association into large crystals depended on the use of alpha-octyl glucoside, a detergent with relatively low affinity for the protein. Electron micrographs of thin-sectioned crystals show a high degree of order. Preliminary crystallographic data suggest that the crystals, which exhibit diffraction to 3.8 A, have a cubic space group.

Bacterial Proteins↗