Repair of impurity-poisoned protein crystal surfaces.

The surface morphology of Bence-Jones protein (BJP) crystals was investigated during growth and dissolution by using in situ atomic force microscopy (AFM). It was shown that over a wide supersaturation range, impurities adsorb on the crystalline surface and ultimately form an impurity adsorption layer that prevents further growth of the crystal. At low undersaturations, this impurity adsorption layer prevents dissolution. At greater undersaturation, dissolution takes place around large particles incorporated into the crystal, leading to etch pits with impurity-free bottoms. On restoration of supersaturation conditions, two-dimensional nucleation takes place on the impurity-free bottoms of these etch pits. After new growth layers fill in the etch pits, they cover the impurity-poisoned top layer of the crystal face. This leads to the resumption of its growth. Formation of an impurity-adsorption layer can explain the termination of growth of macromolecular crystals that has been widely noted. Growth-dissolution-growth cycles could be used to produce larger crystals that otherwise would have stopped growing because of impurity poisoning.

Binding of nascent collagen by amyloidogenic light chains and amyloid fibrillogenesis in monolayers of human fibrocytes.

Light (L) chain dimers expressed by multiple myeloma cells and collected as Bence-Jones proteins from the urine of human subjects were tested for their ability to form deposits in fibroblast monolayer cell cultures. Bence-Jones proteins from subjects with primary amyloidosis associated with L chains were shown to form fibrillar deposits by the in vitro assay introduced in this report. Filaments interspersed with nascent collagen could be detected after only 48 h. Deposition of L chains continued over a period of 72 h culminating in the appearance of dense fibrils with widths of 80-100 A and a variety of lengths. Formation of amyloid-like fibrils was accompanied by interference with the maturation of the collagen produced by the fibroblast cells. Fibrils composed of the Mcg lambda-type L chain were deposited between collagen fibers, thus expanding them laterally and leading to their partial disintegration. Mature collagen was completely missing from fibroblast monolayers exposed to the Sea lambda chain and the Jen kappa chain. Collagen with the characteristic striped pattern matured normally in control samples, such as those not dosed with amyloid precursors or those treated with a non-amyloidogenic Bence-Jones protein (e.g., the Hud lambda chain dimer). By immunochemical techniques using fluorescein- and gold-labeled anti-L chain antibodies, amyloidogenic L chains were shown to decorate the strands of nascent collagen. This observation suggests that amyloidogenic L chains are concentrated in the extracellular matrix by monovalent antigen-antibody type reactions. The capacity of the Mcg L chain dimer to bind collagen-derived sequences was tested by soaking crystals with a collagenase substrate, PZ-Pro-Leu-Gly-Pro-D-Arg. Difference Fourier analysis at 2.7 A resolution indicated that the PZ-peptide is a site-filling ligand. It could not be removed from the active site by perfusion of the crystal with ammonium sulfate crystallizing media. Similar experiments with the collagen-derived peptide (Pro-Pro-Gly)(5) showed substantial hysteresis effects extending from one end of the Mcg dimer to the other. After the ligand was withdrawn, the active site of the Mcg dimer could no longer bind the PZ-peptide. However, if the active site was first blocked by the PZ-peptide and subsequently exposed to the (Pro-Pro-Gly)(5) peptide, the difference Fourier map was indistinguishable from that obtained with the PZ-peptide alone. We concluded that amyloidogenic L chains such as the Mcg dimer could be concentrated in the perivascular space by binding to normal tissue constituents. These components include nascent collagen, which can be deterred from maturing as a result of this binding. Participation in such pathological activity is also self-destructive to the amyloidogenic L chains, which lose their binding capabilities for collagen-derived peptides and also become susceptible to irreversible conversion to amyloid fibrils. All of these events may be prevented by prior treatment of the amyloidogenic L chains with site-filling ligands. (c) 2000 John Wiley & Sons, Ltd.

Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro.

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.

Optimal protein-folding codes from spin-glass theory.

Protein-folding codes embodied in sequence-dependent energy functions can be optimized using spin-glass theory. Optimal folding codes for associative-memory Hamiltonians based on aligned sequences are deduced. A screening method based on these codes correctly recognizes protein structures in the "twilight zone" of sequence identity in the overwhelming majority of cases. Simulated annealing for the optimally encoded Hamiltonian generally leads to qualitatively correct structures.

Three-dimensional structure of a light chain dimer crystallized in water. Conformational flexibility of a molecule in two crystal forms.

The three-dimensional structure of an immunoglobulin light chain dimer (Mcg) crystallized in deionized water (orthorhombic form) was determined at 2.0 A resolution by phase extension and crystallographic refinement. This structure was refined side-by-side with that of the same molecule crystallized in ammonium sulfate (trigonal form). The dimer adopted markedly different structures in the two solvents. "Elbow bend" angles between pseudo 2-fold axes of rotation relating pairs of "variable" (V) and "constant" (C) domains were found to be 132 degrees in the orthorhombic form and 115 degrees in the trigonal form. Modes of association of the V domains and, to a lesser extent, the pairing interactions of the C domains were different in the two structures. Alterations in the V domain pairing were reflected in the shapes of the binding regions and in the orientations of the side-chains lining the walls of the binding sites. In the trigonal form, for instance, the V domain interface was compartmentalized into a main binding cavity and a deep pocket, whereas these spaces were continuous in the orthorhombic structure. Patterns of ordered water molecules were quite distinct in the two crystal types. In some cases, the solvent structures could be correlated with conformational changes in the proteins. For example, close contacts between V and C domains of monomer 1 of the trigonal form were not retained in orthorhombic crystals. Ordered water molecules filled the space created when the two domains moved apart.

Dynamic structures of globular proteins with respect to correlative movements of residues calculated in the normal mode analysis.

Dynamic structures of globular proteins are studied on the basis of correlative movements of residues around their native conformations, which are computed by means of the normal mode analysis. To describe the dynamic structures of a protein, the core regions moving with strong positive or negative correlations to other regions of the polypeptide chain are detected from the correlation maps of the movements of residues. Such core regions are different, according to the definition, from the regions defined from a geometrical point of view, such as secondary structures, domains, modules, and so on. The core regions are actually detected for four proteins, myoglobin, Bence-Jones protein, flavodoxin, and hen egg-white lysozyme, with different folding types from each other. The results show that some of them coincide with the secondary structures, domains, or modules, but others do not. Then, the dynamic structure of each protein is discussed in terms of the dynamic cores detected, as compared with the secondary structures, domains, and modules.