A.Joseph Kalb

The separation of three l-fucose-binding proteins of Lotus tetragonolobus

The l-fucose-binding protein of Lotus tetragonolobus has been separated into three components. Each of the components is an l-fucose-binding protein; however, the three proteins differ in molecular weight, binding constant for l-fucose and chemical composition.

Crystal structure of demetallized concanavalin A: the metal-binding region.

Abstract We have determined the crystal structure of demetallized concanavalin A, at a resolution of 3.2 A, by molecular replacement using the known structure of native concanavalin A. Refinement of the initial model using a constraint-restraint reciprocal-space least-squares procedure caused the conventional crystallographic agreement ( R ) factor to decrease from 0.47 to a final value of 0.26. There are significant conformational changes in the metal-binding region involving residues Asp 19 and His24, which are substantially closer to each other than in native concanavalin A. These residues form an internal salt bridge which does not exist when the metal ions are attached to the protein. The binding site for transitionmetal ions is still intact, but the calcium site is not, since one of its two carboxylic ligands, Asp 19, is unavailable. Flexibility is observed for one of the transitionmetal ligands, Glu8, as well as for some segments of the backbone. The latter could account for the increased susceptibility of demetallizcd concanavalin A to proteolysis.

An X-ray crystallographic study of concanavalin A

Abstract We have studied the X-ray diffraction of single crystals of concanavalin A. The protein crystallizes in space group I222 or I212121 with unit cell dimensions a = 87.2 A , b = 89.2 A , c = 62.9 A . The weight of protein per unit cell is 21 ± 2 × 104 daltons. The asymmetric-unit weight is 2.6 ± 0.3 × 104 daltons. This is one-half the molecular weight of concanavalin A in solution and is equal to the equivalent binding weight of concanavalin A for transition metal ions, calcium ions and α-methyl- d -glucopyranoside.

An x-ray crystallographic study of demetallized concanavalin A.

Abstract Demetallized concanavalin A crystallizes in space group P 2 1 22 1 with unit cell dimensions a = 85.4 A , b = 91.5 A , c = 61.3 A . The asymmetric unit contains one protein molecule. The rotation and translation functions indicate the existence of a local dyad axis some 7 ° off the z -axis with a screw component leading to a relative subunit translation of about 6 A. The demetallized crystals are converted to the native form on soaking in dilute solution of transition metal ions and Ca 2+ ions.

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.

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.

Magnetic resonance relaxation of 1H and 17O in aqueous solutions of concanavalin A

Abstract We have studied the nuclear magnetic resonance relaxation times of 1 H and 17 O nuclei in aqueous solutions of Mn 2+ complexes of concanavalin A. The results indicate that the Mn 2+ is coordinated to one exchangeable water molecule. The mean residence time, τ h , of this water molecule at the Mn 2+ is approximately 1 · 10 −7 s in Mn 2+ -concanavalin A and is ten times as long in Mn 2+ , Ca 2+ -concanavalin A. No changes occur on binding of α-methyl glucoside, or on raising the pH to a value where molecular aggregation occurs. It is possible that on occupation of the Ca 2+ -binding site, the immediate electronic environment of the Mn 2+ alters and thus affects the mean residence time of the water molecule. It is also possible that the increase in τ h is brought about by restrictions imposed on the exchange of water molecules between bulk solvent and the Mn 2+ .

Visible absorption and circular dichroism of the cobalt complexes of concanavalin A

Abstract The visible and CD spectra of the cobalt complexes of concanavalin A have been determined. The visible spectra of Co 2+ -concanavalin A, Co 2+ , Ca 2+ -concanavalin A and the α-methyl glucoside complex of Co 2+ , Ca 2+ -concanavalin A are identical and are characterized by a broad region of absorption with a maximum at 515 nm ( ϵ 515 nm = 33 cm −1 ·l·gatom −1 ) and shoulders at 490 nm and 470 nm. The CD of Co 2+ -concanavalin A consists of at least four positive transitions between 400 and 600 nm. The optical anisotropy is as high as +0.15. When Ca 2+ is bound, two of the CD transitions disappear. No further change occurs on binding of α-methyl glucoside. These findings suggest that the transition metal binding site in concanavalin A has nearly octahedral symmetry with some deviation from centrosymmetry. This deviation is decreased on binding of Ca 2+ to the protein.

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

A saccharide ligand on the outer surface of retinal rod disc membranes

Abstract Binding of concanavalin A to bleached bovine retinal rod disc membranes is governed by a single association constant of 6.4 × 10 7 M −1 , at 26 °C. α-Methyl glucoside competes with the saccharide ligand of the membrane for the saccharide-binding site of concanavalin A. The surface density of the saccharide ligand is calculated to be one ligand per 5 × 10 4 A 2 of membrane surface.