How ionic liquids can help to stabilize native proteins

The native state of a globular protein is essential for its biocatalytic function, but is marginally stable against unfolding. While unfolding equilibria are often reversible, folding intermediates and misfolds can promote irreversible protein aggregation into amorphous precipitates or highly ordered amyloid states. Addition of ionic liquids—low-melting organic salts—offers intriguing prospects for stabilizing native proteins and their enzymatic function against these deactivating reaction channels. The huge number of cations and anions that form ionic liquids allows fine-tuning of their solvent properties, which offers robust and efficient strategies for solvent optimization. Going beyond case-by-case studies, this article aims at discussing principles for a rational design of ionic liquid-based formulations in protein chemistry and biocatalysis

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzymeindependent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmocsolidphase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with FmocSPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the Cterminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified fulllength domain.(VLID)310652

The Journal of Biological Chemistry / Structural analyses of Arabidopsis thaliana legumain reveal differential recognition and processing of proteolysis and ligation substrates

Legumain is a dual-function proteasepeptide ligase whose activities are of great interest to researchers studying plant physiology and to biotechnological applications. However, the molecular mechanisms determining the specificities for proteolysis and ligation are unclear because structural information on the substrate recognition by a fully activated plant legumain is unavailable. Here, we present the X-ray structure of Arabidopsis thaliana legumain isoform (AtLEG) in complex with the covalent peptidic Ac-YVAD chloromethyl ketone (CMK) inhibitor targeting the catalytic cysteine. Mapping of the specificity pockets preceding the substrate-cleavage site explained the known substrate preference. The comparison of inhibited and free AtLEG structures disclosed a substrate-induced disorderorder transition with synergistic rearrangements in the substrate-recognition sites. Docking and in vitro studies with an AtLEG ligase substrate, sunflower trypsin inhibitor (SFTI), revealed a canonical, protease substratelike binding to the active sitebinding pockets preceding and following the cleavage site. We found the interaction of the second residue after the scissile bond, P2′S2′, to be critical for deciding on proteolysis versus cyclization. cis-trans-Isomerization of the cyclic peptide product triggered its release from the AtLEG active site and prevented inadvertent cleavage. The presented integrative mechanisms of proteolysis and ligation (transpeptidation) explain the interdependence of legumain and its preferred substrates and provide a rational framework for engineering optimized proteases, ligases, and substrates.W_01213M1901(VLID)266778

The recombinant inhibitor of DNA binding Id2 forms multimeric structures via the helix-loop-helix domain and the nuclear export signal

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to -mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.(VLID)253307

The Modern Face of Synthetic Heterocyclic Chemistry

The synthesis of heterocycles is arguably one of the oldest and at the same time one of the youngest disciplines of organic chemistry. Groundbreaking principles to form heterocycles, mainly by condensation reactions, were recognized in the beginning of the 19th century, and many of the classical reactions discovered at that time are still of great value today. In the 21st century, the wealth of synthetic methodology toward heterocycles is overwhelming, and catalysis, in particular, as one of the cornerstones of green and sustainable chemistry has contributed in a major way to these developments. This perspective tries the impossible by discussing some recent advances in the construction of heterocycles, focusing on catalytic methodology. We are aware that we do not come close to giving adequate credit to the great creativity of chemists in the field

Novel Strategies for the Synthesis of Peptides containing Cis- or Trans-β-Aminocyclopropyl Carboxylic Acids

The scope and limitation of a new in situ coupling strategy is described, which allows the incorporation of cis- or trans-β-aminocyclopropanecarboxylic acids into peptides