Joseph Yariv

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha‐D‐mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X‐ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide‐free concanavalin A. The structure of the saccharide complex was refined by restrained least‐squares methods to an R‐factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha‐D‐mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium‐binding site and 12.8 A from the transition metal‐binding site. A network of seven hydrogen bonds connects oxygen atoms O‐3, O‐4, O‐5 and O‐6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O‐2 and O‐1 of the mannoside extend into the solvent. O‐2 is hydrogen‐bonded through a water molecule to an adjacent asymmetric unit. O‐1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.

The structure of concanavalin A and its bound solvent determined with small-molecule accuracy at 0.94 [Aring ]resolution

The structure of concanavalin A, a saccharide-binding metalloprotein of subunit molecular weight 25000 Da§, is reported with precision and accuracy equivalent to those of small-molecule crystal structures. An X-ray data-to-parameter ratio of 6.1:1 has allowed the first unrestrained anisotropic refinement of a protein structure and, indeed, one of large molecular weight. This has been achieved by the combined power of synchrotron radiation, a highly sensitive CCD-based area detector and cryogenic techniques on crystals of exceptional quality. The high precision of the atomic coordinates and the direct observation of many hydrogen atom positions, on both the protein and bound solvent, provide new insights into the protein structure, including precise geometry of the metal-binding sites, the role of water molecules in the extended saccharide-binding site, the structure and dynamics of bound and buried water, and direct evidence that proximal carboxylic acid side chains can exist as carboxyl–carboxylate pairs. The biophysical chemistry of this protein is thereby elucidated in detail in this and the companion paper.

X-Ray and molecular dynamics studies of concanavalin-A glucoside and mannoside complexes Relating structure to thermodynamics of binding

Crystallographic and computational methods have been used to study the binding of two monosaccharides (glucoside and mannoside) to concanavalin-A. The 2 A structure of glucoside bound concanavalin-A is reported and compared with the 2 A structure of the mannoside complex. The interaction energies of the substrate in each crystallographic subunit were calculated by molecular mechanics and found to be essentially the same for both sugars. Further energy minimisation of the active site region of the subunits did not alter this conclusion. Information from crystallographic B-factors was interpreted in terms of mobility of the sugars in the combining site. Molecular dynamics (MD) was employed to investigate mobility of the ligands at the binding sites. Switching between different binding states was observed for mannoside over the ensemble in line with the crystallographic B-factors. A calculated average interaction energy was found to be more favourable for mannoside than glucoside, by 4.9±3.6 kcal mol-1 (comparable with the experimentally determined binding energy difference of 1.6±0.3 kcal mol-1). However, on consideration of all terms contributing to the binding enthalpy a difference is not found. This work demonstrates the difficulty in relating structure to thermodynamic properties, but suggests that dynamic models are needed to provide a more complete picture of ligand–receptor interactions.

Mössbauer spectroscopy of Escherichia coli and its iron-storage protein

57Fe Mössbauer spectra of whole frozen Escherichia coli cells and of an iron storage protein isolated from iron-rich cells of E. coli have been measured over a range of temperatures down to 0.08 K. The spectra of E. coli cells with high iron content and of the iron storage protein were found to be very similar. Above 4 K these spectra consist of a quadrupole split doublet characteristic of Fe3+. Below 3.5 K, the spectra display magnetic hyperfine splitting which is temperature dependent, and point to the existence of an ordered magnetic phase associated with a saturation magnetic hyperfine field of 43 tesla in both samples. The results indicate that the bulk of iron in the iron-rich cells is in the form of aggregates similar in nature to the iron cores in the isolated protein, although the latter account for not more than 1% of the total iron in the cells. The Mössbauer spectra of the isolated protein are different from those observed in ferritin, the iron-storage protein of plants and higher animals, showing that the iron cores in these two proteins are different.

The use of I-amino-L-fucose bbound to sepharose in the isolation of L-fucose-binding proteins

Abstract N-(ϵ- Aminocaproyl )-β- L - fucopyranossylamine was synthesized and linked covalently to an agarose resin. The modified resin binds specifically the three L -fucose-binding proteins present in Lotus tetragonolobus seeds and was used for their isolation by an affinity chromatographic procedure.

Electron spin resonance study of the transition metal-binding site of concanavalin A

Abstract The ESR spectrum of Mn 2+ bound to the transition metal-binding site of concanavalin A is characteristic of bound Mn 2+ with low coordination symmetry. When Ca 2+ is bound to the Ca 2+ -binding site of concanavalin A, the spectrum of bound Mn 2+ is modified. Binding of methyl-α- d -glucopyranoside to the saccharide-binding site causes no change in the spectrum.

Labelling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine

A’-Bromoacetyl P-D-galactopyranosylamine was designed as an affinity label for Zac-permease [l] which has a reactive sulphydryl group at its substrate binding site [2] . The observed irreversible inactivation of the active transport of lac-permease substrates by this reagent is, however, not site-specific since active transport mediated by another permease, a MG-permease, is also inactivated [3] . In the course of our investigations of kc-permease, this reagent was found to inactivate /3-galactosidase in intact E. coli cells [4] . We describe here the inactivation of purified fl-galactosidase by N-bromoacetyl&D-galactopyranosylamine. One mole of reagent is bound to the enzyme per mole of site inactivated. The described labelling procedure should facilitate the study of the peptide sequence in the vicinity of the active site of this enzyme.

Molecular size and symmetry of the bacterioferritin of Escherichia coli: X-ray crystallographic characterization of four crystal forms

X-ray crystallographic data from four crystal forms of Escherichia coli bacterioferritin show that the molecule has a diameter in the range 119 to 128 A. Molecules are composed of 24 subunits arranged in 432 symmetry. In both size and symmetry the molecule resembles ferritin from eukaryotes. The four crystal forms are monoclinic, space group P2(1) with unit cell dimensions a = 118.7 A, b = 211.6 A, c = 123.3 A and beta = 119.1 degrees; orthorhombic, C222(1), a = 128.7 A, b = 197.1 A, c = 202.8 A; tetragonal, P4(2)2(1)2, a = b = 210.6 A, c = 145.0 A and cubic, I432, a = 146.9 A.

Hydrolysis of alanine oligopeptides by an enzyme located in the membrane of Mycoplasma laidlawii

Abstract Membranes obtained from Mycoplasma laidlawii and whole cells suspended in an isotonic salt solution cleva the N(α)- termional l -alanine residue of oligoalanine peptides of the general formula Ala( l )-[Ala( l )-Ala( d )]n, n = 2,4,8 . The intact cells were found to be impermeable to l -alaine when suspended in an isotonic salt solution Entry of l -alanine into the cells was observed only whens suspended in the appropriate growth medium.

Two locations of the lac permease sulphydryl in the membrane of E. coli

Lzc Permease is a protein involved in the active transport of lactose, and is located in the membrane of E. coli [l ,2] . The lac permease molecule has a sulphydryl group which is essential for transport [3,4] . The mechanism by which permease facilitates the transport of lactose into the cell is not known. One of the possibilities is that permease serves as a vehicle which physically moves the substrate through a permeability barrier. If this is so, permease must exist in the membrane in at least two states: one in which the binding site is on one side of a permeability barrier, and another in which the site is on the other side of the barrier. In this work we have used mercurials (PMBS and and P-Hg)** to inactivate lac permease and sulphydry1 compounds (mercaptoethanol and PSH) to reactivate it. Permease inactivated either by PMBS or by P-Hg can be fully reactivated by mercaptoethanol. A difference between PMBS inactivated and PHg inactivated permease is observed when reactivation by P-SH is attempted: P-Hg inactivated permease is fully reactivated whereas PMBS inactivated permease is only partially reactivated. These findings strongly suggest the existence of