Light-chain (L-chaln) amyloldoss Is characterzed by depit of Nbrillar aregtes composed of the N-

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It is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.Keywords: complementarity determining regions; hypervariable loops; immunoglobulin stability; protein stability; Stern-Volmer plots; unfolding In the structures of antibody heavy and light chain variable domains, complementarity determining region (CDR) loops of diverse sequence and conformation project from P-sandwich frameworks to determine the affinity and specificity of antigen binding. These diverse sequences arise in the development of an antibody response via a complex combination of gene rearrangements and somatic mutations (Branden & Tooze, 1991). Protein engineers have transferred antigen binding activity from one framework to another by swapping hypervariable CDR loops between frameworks (Jones et al., 1986;Riechmann et al., 1988). In addition, totally artificial loop sequences have been introduced into variable domain frameworks to generate novel binding functions (Barbas et al., 1993;Fisch et al., 1994). This apparent tolerance of the immunoglobulin fold for both sequence diversity and loop swaps is consistent with the view that the major determinants of structural stability of globular proteins lie in the sequence-specific formation and packing of regular secondary structure elements (Shortle, 1992;Matthews, 1993), whereas surface loops are more forgiving of sequence changes (EL Hawrani et al., 1994).Although the CDR loops differ greatly in sequence, they are not infinitely variable in either sequence or conformation. Positions with highly conserved amino acids are found within many CDR sequences (Kabat et al., 1991 of "canonical structures" appear to be available for each CDR. For example, in an examination of the structural database available in 1989, only four different loop conformations were identified in the light chain crystallographic database for CDRl, and only three for CDR3 . If these patterns d o suggest constraints on the sequence variability of CDRs, however, it remains difficult from examination of evolved antibodies to either gauge the severity of these constraints or to understand their structural or functional basis.In this report we describe the preparation of a number of mutants of the immunoglobulin light chain variable domain REI, in which wild-type CDR sequences were replaced by loops of differing sequence and length. These replacements cause dramat...

The results implicate a late, equilibrium folding intermediate in IB formation, in contrast to the apparent involvement of transient folding intermediates in other IB systems described to date. As equilibrium unfolding intermediates have also been implicated in light chain amyloidosis and deposition diseases, IB formation may prove a useful model for these human diseases.

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The nucleotide sequence encoding the structural gene (651 bp) and flanking regions for the assimilatory-type sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) was determined after cloning a 1.4 kb HindIII/SalI genomic fragment possessing the gene into Bluescript pBS(+)KS. The primary structure of the protein was deduced, and the molecular mass of the apoprotein was estimated as 24 kDa. The amino acid sequence of the polypeptide shows some similarities at putative [Fe4S4] cluster binding sites in comparison with the heme protein subunit of the larger Escherichia coli and Salmonella typhimurium sulfite reductases and spinach nitrite reductase. This is the first reported sequence of a member of a new class of low molecular weight assimilatory sulfite-reducing enzymes recently identified in a number of anaerobic bacteria [Moura, I., Lina, A. R., Moura, J. J. G., Xavier, A. V., Fauque, G., Peck, H. D., & Le Gall, J. (1986) Biochem. Biophys. Res. Commun. 141, 1032-1041].

We describe a structural validation of the use of presentation scaffolds for control and elucidation of bioactive conformations of peptides. The protein REI-RGD34--produced by inserting the sequence RIPRGDMP into the CDR1 loop region of the immunoglobulin VL domain REI--strongly inhibits fibrinogen binding to the integrins alpha IIb beta 3 and alpha V beta 3. In the X-ray crystal structure of their protein at 2.4 A resolution, the RGD-containing loop exhibits defined electron density that is consistent with models for the bioactive conformations of ligands of these receptors based on previous small-molecule studies. Furthermore, a search of a small-molecule database with conformational information derived from the structure of REI-RGD34 identified constrained peptides and peptidomimetics known to be antagonists of the platelet receptor alpha IIb beta 3.

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