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Biomedical subjects

A Yonath

Publications and source records attributed to A Yonath.

71 records · Page 4Linked to original sources
PubMed1977

Structural analysis of denaturant-protein interactions: comparison between the effects of bromoethanol and SDS on denaturation and renaturation of triclinic lysozyme.

This paper summarizes our crystallographic studies of the interaction of denaturants with cross-linked triclinic lysozyme. Electron density maps of various bromoethanol-lysozyme complexes are analyzed and compared to those reported earlier for SDS-lysozyme complexes. Despite differences in the chemical nature and size of the two denaturants their mode of interaction with the protein is quite similar, suggesting the existence of a general mechanism for binding of hydrophobic-hydrophilic denaturants to proteins. Our results are consistent with the conclusion that lysozyme consists of two domains connected by a flexible segment and that this segment represents an internal degree of freedom of the protein.

Ethanol↗
PubMed1977

Crystallographic studies of protein denaturation and renaturation. 1. Effects of denaturants on volume and X-ray pattern of cross-linked triclinic lysozyme crystals.

Triclinic crystals of hen egg-white lysozyme cross-linked with glutaraldehyde have been treated with various denaturants and found to be susceptible to x-ray structure analysis even after major conformational changes in the protein. Cross-linked crystals were isomorphous with the native form, and electron density difference maps indicated the locations of intermolecular corss-links, but showed no appreciable differences in the protein conformation. Soaking of the cross-linked crystals in danaturant solutions of increasing concentrations caused corresponding increases in crystal volume and decreases in minimum observable x-ray spacings. These changes proved partly reversible on diluting the solutions, and measurements of crystal volume and minimums x-ray spacing were used to follow denaturation and renaturation as a function of concentration for several denaturants. Some of these, including bromoethanol and sodium dodecyl sulfate, had little effect on the crystals below critical concentrations at which there was a sharp volume increase and loss of x-ray pattern, which could, however, be regenerated to about 3.2-A resolution. Others, including KCNS and urea, caused more gradual changes, but with a smaller degree of recovery. It is suggested that at least two different denaturation mechanisms are involved with detergent-like reagents disrupting the hydrophobic interactions joining the two wings of the lysozyme molecule and hydrophilic denaturants interacting primarily with polar groups on the molecular surface.

Amino Acids↗
PubMed1977

Crystallographic studies of protein denaturation and renaturation. 2. Sodium dodecyl sulfate induced structural changes in triclinic lysozyme.

Cross-linked triclinic lysozyme was denatured with sodium dodecyl sulfate. Removal of the denaturant resulted in a refolding of the protein to a conformation similar to but not identical with the native one. Three-dimensional x-ray diffraction data out to 3.2-A resolution were collected for two states in the refolding pathway, and appropriately weighted electron density difference maps were constructed. An analysis of these maps reveals that a sodium dodecyl sulfate molecule is trapped in the interior of the protein, and results in a separation of regions of the polypeptide chain. Our results are discussed in terms of current models for protein folding.

Binding Sites↗
PubMedDate not supplied

The interplay between X-ray crystallography, neutron diffraction, image reconstruction, organo-metallic chemistry and biochemistry in structural studies of ribosomes.

Crystals of ribosomes, their complexes with components of protein biosynthesis, their natural, mutated and modified subunits, have been subjected to X-ray and neutron crystallographic analyses. Electron microscopy and 3-dimensional image reconstruction, supported by biochemistry, genetic, functional and organo-metallic studies were employed for facilitating phasing of the crystallographic data. For example, a monofunctional multi heavy-atom cluster (undecagold) was designed for covalent and quantitative binding to ribosomes. The modified particles were crystallized isomorphously with the native ones. Their difference-Patterson maps contain indications for the usefulness of these derivatives for subsequent phasing. Models of the ribosome and its large subunit were reconstructed from tilt series of 2-dimensional sheets. The comparison of the various reconstructed images enabled an initial assessment of the reliability of these models and led to tentative assignments of several functional features. These include the presumed sites for binding mRNA and for codon-anticodon interactions, the path taken by the nascent protein chain and the mode for tRNA binding to ribosomes. These assignments assisted in the design of biologically meaningful crystal systems. The reconstructed models are being used to identify structural features in initial density maps derived from X-ray and neutron diffraction data.

Crystallography↗
PubMedDate not supplied

Crystallography of ribosomes: attempts at decorating the ribosomal surface.

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

Bacteria↗