A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye, Thioflavin-T (ThT), has for decades been used in the diagnosis of protein-misfolding diseases and also in kinetic studies of selfassembly (fibrillization). Despite its importance, efforts to characterize the ThT binding mechanism at an atomic level have been hampered by the inherent insolubility and heterogeneity of peptide selfassemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic" (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of β-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM β-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands ("cross-strand ladders") that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThTbinding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. Their x-ray crystal structures suggest that ThT interacts with the β-sheet by docking on surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more β-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.Keywords amyloid fibrils; cross-β; β-sheet; tyrosine; protein engineeringThe formation of β-rich peptide self-assemblies is associated with a diverse set of the so-called protein misfolding diseases. 1; 2 Accordingly, there have been intense interest and research activities in the development of small organic molecules for the detection and inhibition of fibril formation. 3-7 Although it is clear that understanding how these small molecules interact with fibrils would greatly aid the development of effective diagnostic and therapeutic reagents, there is a paucity of experimental data that provide such information at an atomic level. Fibrils Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. and other peptide self-assemblies are insoluble and often heterogeneous in their composition, making it extremely difficult to characterize their interaction...

Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting ''affinity clamp'' had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution.affinity reagent ͉ epitope ͉ molecular evolution ͉ protein design ͉ PDZ domain D irected evolution-based protein engineering creates new protein functions by exploiting processes that occur during natural evolution of proteins. Protein evolution progresses via point mutations, duplication, and recombination of genes under selective pressure. The processes of gene duplication and subsequent sequence divergence (1) have been successfully recapitulated in directed evolution and computational protein design (2, 3) where preexisting active sites within natural protein scaffolds are altered to produce new functions (Fig.

Although the ␤-rich self-assemblies are a major structural class for polypeptides and the focus of intense research, little is known about their atomic structures and dynamics due to their insoluble and noncrystalline nature. We developed a protein engineering strategy that captures a self-assembly segment in a water-soluble molecule. A predefined number of self-assembling peptide units are linked, and the ␤-sheet ends are capped to prevent aggregation, which yields a mono-dispersed soluble protein. We tested this strategy by using Borrelia outer surface protein (OspA) whose single-layer ␤-sheet located between two globular domains consists of two ␤-hairpin units and thus can be considered as a prototype of self-assembly. We constructed self-assembly mimics of different sizes and determined their atomic structures using x-ray crystallography and NMR spectroscopy. Highly regular ␤-sheet geometries were maintained in these structures, and peptide units had a nearly identical conformation, supporting the concept that a peptide in the regular ␤-geometry is primed for self-assembly. However, we found small but significant differences in the relative orientation between adjacent peptide units in terms of ␤-sheet twist and bend, suggesting their inherent flexibility. Modeling shows how this conformational diversity, when propagated over a large number of peptide units, can lead to a substantial degree of nanoscale polymorphism of self-assemblies.␤-sheet ͉ ␤-strand interaction ͉ amyloid fibril ͉ nanomaterial ͉ protein engineering

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine antihuman epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1 ⁄ 40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.Structural and functional analyses of antigen-antibody protein-protein interactions have revealed that complementaritydetermining regions (CDRs) 5 in the variable domains of antibodies play a critical role in the specificity and affinity of the antibodies for their targets by means of shape and charge complementarities (1-4). Antibodies have a common fold (the immunoglobulin fold), and hypervariable CDRs are located on one edge of the framework region (1-4). Grafting of the CDRs of antibodies onto the frameworks of other antibodies has been attempted (5-9). Especially interesting from diagnostic and therapeutic viewpoints is the humanization, or reshaping, of murine antibodies, whereby a set of CDRs from murine antibodies are transplanted to appropriate scaffolds of human antibodies to reduce immune responses against murine antibodies in human hosts (10 -20).However, grafting of the six CDRs of murine antibodies onto appropriate frameworks of human antibodies often results in reduced affinity or specificity for the target antigen (10, 14, 16). The pioneering work of Foote and Winter (21) has suggested that antibody residues in th...

Purpose: Bispecific antibodies (BsAb) have been exploited as both cancer immunodiagnostics and cancer therapeutics and show promise in clinical trials of cancer imaging and therapy. For development of BsAbs as clinical reagents, we have focused on construction of small recombinant BsAbs, called bispecific diabodies. Here, we constructed and characterized a humanized bispecific diabody. Experimental Design: We have reported significant antitumor activity of an anti-epidermal growth factor receptor (EGFR) Â anti-CD3 bispecific diabody (Ex3) in in vitro cytotoxicity assays and in vivo. We humanized the Ex3 diabody (hEx3) by grafting the complementaritydetermining region and compared its biological properties with those of Ex3. We also tested its physiologic stability and ability to alter survival in xenografted mice. Results: The final yield of hEx3 was 10 times that of Ex3, and refolded hEx3 and Ex3 showed identical binding profiles in EGFR-positive cell lines and EGFR-transfected Chinese hamster ovary cells. hEx3 showed dose-dependent cytotoxicity to EGFR-positive cell lines, which could be specifically inhibited by parental monoclonal antibody IgGs against EGFR or CD3 antigens. The heterodimeric structure was retained in PBS for 6 months, and growth inhibition was maintained after incubation under physiologic conditions. Coadministration of hEx3 with T-LAK cells and interleukin-2 prolonged the survival of nude mice with human colon carcinoma. Conclusions:The humanized diabody hEx3 is an attractive molecule for cancer therapy and may provide important insights into the development of EGFR-based cancer-targeting reagents.