De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing.

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

The reasons underlying the oligomeric nature of some proteins such as triosephosphate isomerase (TIM) are unclear. It has been proposed that this enzyme is an oligomer, mainly because of its stability rather than for functional reasons. To address this issue, the reversible denaturation and renaturation of the homodimeric TIM from baker's yeast ( Saccharomyces cerevisiae ) induced by guanidinium chloride and urea have been characterized by spectroscopic, functional and hydrodynamic techniques. The unfolding and refolding of this enzyme are not coincident after 'conventional' equilibrium times. Unfolding experiments did not reach equilibrium, owing to a very slow dissociation and/or unfolding process. By contrast, equilibrium was reached in the refolding direction. The simplest equilibrium pathway compatible with the obtained data was found to be a three-state process involving an inactive and expanded monomer. The Gibbs energy changes for monomer folding (delta G (0)(fold) = -16.6+/-0.7 kJ x mol(-1)) and monomer association (delta G (0)(assoc) = -70.3+/-1.1 kJ x mol(-1)) were calculated from data obtained in the two denaturants. From an analysis of the present data and data from the literature on the stability of TIM from different species and for other beta/alpha barrels, and model simulations on the effect of stability in the catalytic activity of the enzyme, it is concluded that the low stability of the monomers is neither the only, nor the main, cause for the dimeric nature of TIM. There is interplay between function and stability.

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Transient protein–protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein–protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine βlg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. ΔH and ΔCp were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near‐UV CD spectra proved to be independent of protein concentration, indicating a rigid body‐like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental ΔCp. Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces. Proteins 2008. © 2007 Wiley‐Liss, Inc.

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Approximately 25% of the k6 light chains have glycine rather than arginine at position 25, which is an allelic variant of the IGLV6-57 (6a) locus. The Gly25 variant has been shown to decrease the folding stability of the germline k6 V L protein 6aJL2 by 1.7 kcalÁmol À1. In this work, we compared the thermodynamic and fibrillogenic properties of the amyloidosis (AL) derived recombinant (r) V L protein AR, which contains the allelic variant Gly25, with those of germline rV L 6aJL2-R25G and the k6 diseaseassociated V L proteins Wil (AL) and Jto (myeloma). Our experiments show that of the four proteins AR is the least stable; forms amyloid fibrils at physiological temperature, pH and ionic strength; has the shortest lag time; and elongates homologous seeds most efficiently. We conclude that the Gly25 allelic variant, together with the somatic mutations, contributes importantly to the extremely low stability and high amyloidogenicity of the AL-derived protein AR.