Random Amino Acid Mutations and Protein Misfolding Lead to Shannon Limit in Sequence-Structure Communication

The transmission of genomic information from coding sequence to protein structure during protein synthesis is subject to stochastic errors. To analyze transmission limits in the presence of spurious errors, Shannon's noisy channel theorem is applied to a communication channel between amino acid sequences and their structures established from a large-scale statistical analysis of protein atomic coordinates. While Shannon's theorem confirms that in close to native conformations information is transmitted with limited error probability, additional random errors in sequence (amino acid substitutions) and in structure (structural defects) trigger a decrease in communication capacity toward a Shannon limit at 0.010 bits per amino acid symbol at which communication breaks down. In several controls, simulated error rates above a critical threshold and models of unfolded structures always produce capacities below this limiting value. Thus an essential biological system can be realistically modeled as a digital communication channel that is (a) sensitive to random errors and (b) restricted by a Shannon error limit. This forms a novel basis for predictions consistent with observed rates of defective ribosomal products during protein synthesis, and with the estimated excess of mutual information in protein contact potentials

Thermal Stabilization of an Endoglucanase by Cyclization

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins

High Resolution NMR-based Model for the Structure of a scFv-IL-1β Complex: POTENTIAL FOR NMR AS A KEY TOOL IN THERAPEUTIC ANTIBODY DESIGN AND DEVELOPMENT*

Monoclonal antibodies have recently started to deliver on their promise as highly specific and active drugs; however, a more effective, knowledge-based approach to the selection, design, and optimization of potential therapeutic antibodies is currently limited by the surprising lack of detailed structural information for complexes formed with target proteins. Here we show that complexes formed with minimal antigen binding single chain variable fragments (scFv) reliably reflect all the features of the binding interface present in larger Fab fragments, which are commonly used as therapeutics, and report the development of a robust, reliable, and relatively rapid approach to the determination of high resolution models for scFv-target protein complexes. This NMR spectroscopy-based approach combines experimental determination of the interaction surfaces and relative orientations of the scFv and target protein, with NMR restraint-driven, semiflexible docking of the proteins to produce a reliable and highly informative model of the complex. Experience with scFvs and Fabs targeted at a number of secreted regulatory proteins suggests that the approach will be applicable to many therapeutic antibodies targeted at proteins, and its application is illustrated for a potential therapeutic antibody targeted at the cytokine IL-1β. The detailed structural information that can be obtained by this approach has the potential to have a major impact on the rational design and development of an increasingly important class of biological pharmaceuticals