Rosemarie Raffen

Inhibition of Amyloid Fiber Assembly by Both BiP and Its Target Peptide

Immunoglobulin light chain (LC) normally is a soluble, secreted protein, but some LC assemble into ordered fibrils whose deposition in tissues results in amyloidosis and organ failure. Here we reconstitute fibril formation in vitro and show that preformed fibrils can nucleate polymerization of soluble LC. This prion-like behavior has important physiological implications, since somatic mutations generate multiple related LC sequences. Furthermore, we demonstrate that fibril formation in vitro and aggregation of whole LC within cells are inhibited by BiP and by a synthetic peptide that is identical to a major LC binding site for BiP. We propose that LC form fibrils via an interprotein loop swap and that the underlying conformational change should be amenable to drug therapy.

Increasing Protein Stability by Polar Surface Residues: Domain-Wide Consequences of Interactions Within a Loop

We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the proteins function.

Amyloid fibril formation in microwell plates for screening of inhibitors

Fibril formation is the basis of amyloid production in a number of disease states, such as Alzheimers disease, diabetes and immunocytic dyscrasias. Compounds that inhibit fibril formation could be directly relevant to the treatment of amyloid diseases, and may also provide a foundation for the development of interventions in other molecular condensation diseases ranging from sickle cell anemia to atherosclerosis. We developed an economical and convenient high-throughput method for screening compounds against fibril formation in microwell plates. Chalcones, Jlavonoids and bijluvonoids were screened against fibril formation by a recombinant antibody variable domain (VL). Chalcones 6 and 14 were found to demonstrate inhibition at 0.1 M in 79 M of protein solution in both test tube and microwell plate assays. The concentration of protein in the microwell plate assay could be as low as 5 M using ThTas a monitoring agent. Molecular modeling studies indicated that both compounds could be individually docked into a binding site at the monomer-monomer interface of the VLprotein dimer. These studies suggested that these compounds could potentialty stabilize the VL dimer and therefore reduce its fendency to form fibrils. These findings may provide the basis for a new therapeutic approach to prevent or treat amyloid diseases.

Factors contributing to decreased protein stability when aspartic acid residues are in β-sheet regions

Asp residues are significantly under represented in β‐sheet regions of proteins, especially in the middle of β‐strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a β‐domain. Two Gln residues of the immunoglobulin light‐chain variable domain (VL) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a β‐strand, and that of Q89D, located in the middle of a β‐strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild‐type VL‐Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, VL‐Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild‐type VL‐Len domain. The structures of mutants VL‐Len Q38D and VL‐Len Q89D/M4L/Y27dD/T94H were determined by X‐ray diffraction at 1.6 Å resolution. We found no major perturbances in the structures of these Q→D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main‐chain dihedral angles and in side‐chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a β‐sheet of an immunoglobulin‐type domain can have very serious consequences. A somatic mutation of a β‐strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.

Small zone, high-speed gel filtration chromatography to detect protein aggregation associated with light chain pathologies.

Small zone gel filtration chromatography can be used for qualitative and quantitative analysis of protein interactions and aggregation phenomena. The technique is fast, accessible to most laboratories, and can be combined with computer simulation to extract quantitative information from experimental data. The programs KRUNCH and SCIMZ will be furnished on written request to the authors.