Yurii S. Moroz

Short peptides self-assemble to produce catalytic amyloids

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (> 10,000 Dalton). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of 7-residue peptides that act as Zn2+-dependent esterases. Zn2+ helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. These results indicate that prion-like fibrils are able to not only catalyze their own formation – they also can catalyze chemical reactions. Thus, they might have served as intermediates in the evolution of modern-day enzymes. These results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions.

One-Pot Synthesis of a New Magnetically Coupled Heterometallic Cu2Mn2 [2 × 2] Molecular Grid

The heterometallic [2 x 2] grid-type complex [Cu(2)Mn(2)(pop)(4)(OAc)(4)].7H(2)O (1) has been selectively synthesized in a targeted one-pot reaction. Single-crystal X-ray analysis shows the expected structure with identical metal ions located at diagonal vertices of the grid. Magnetochemical studies reveal that 1 has a ferrimagnetic spin ground state with some admixture of low-lying excited states.

Isolated Toll-like Receptor Transmembrane Domains Are Capable of Oligomerization

Toll-like receptors (TLRs) act as the first line of defense against bacterial and viral pathogens by initiating critical defense signals upon dimer activation. The contribution of the transmembrane domain in the dimerization and signaling process has heretofore been overlooked in favor of the extracellular and intracellular domains. As mounting evidence suggests that the transmembrane domain is a critical region in several protein families, we hypothesized that this was also the case for Toll-like receptors. Using a combined biochemical and biophysical approach, we investigated the ability of isolated Toll-like receptor transmembrane domains to interact independently of extracellular domain dimerization. Our results showed that the transmembrane domains had a preference for the native dimer partners in bacterial membranes for the entire receptor family. All TLR transmembrane domains showed strong homotypic interaction potential. The TLR2 transmembrane domain demonstrated strong heterotypic interactions in bacterial membranes with its known interaction partners, TLR1 and TLR6, as well as with a proposed interaction partner, TLR10, but not with TLR4, TLR5, or unrelated transmembrane receptors providing evidence for the specificity of TLR2 transmembrane domain interactions. Peptides for the transmembrane domains of TLR1, TLR2, and TLR6 were synthesized to further study this subfamily of receptors. These peptides validated the heterotypic interactions seen in bacterial membranes and demonstrated that the TLR2 transmembrane domain had moderately strong interactions with both TLR1 and TLR6. Combined, these results suggest a role for the transmembrane domain in Toll-like receptor oligomerization and as such, may be a novel target for further investigation of new therapeutic treatments of Toll-like receptor mediated diseases.

New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

A Single Mutation in a Regulatory Protein Produces Evolvable Allosterically Regulated Catalyst of Nonnatural Reaction

It only takes one mutation: a strategically placed single mutation in a non-enzymatic protein scaffold produced AlleyCat, a small, allosterically regulated catalyst of Kemp elimination. In only 7 rounds of directed evolution enzymatic efficiency of the original 74 amino acid residue catalyst was improved more than 220-fold to achieve kcat value higher than that of catalytic antibodies for the same reaction, still preserving allosteric regulation.

2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

Design of an allosterically regulated retroaldolase

We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000‐fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatRs activity is allosterically regulated by Ca2+ ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000‐fold) and the change in the apparent pKa of the active lysine residue.

Sulfonyl Fluorides as Alternative to Sulfonyl Chlorides in Parallel Synthesis of Aliphatic Sulfonamides

Two types of aliphatic sulfonyl halides (Cl versus F) were compared in parallel synthesis of sulfonamides derived from aliphatic amines. Aliphatic sulfonyl fluorides showed good results with amines bearing an additional functionality, while the corresponding chlorides failed. Both sulfonyl halides were effective in the reactions with amines having an easily accessible amino group. Aliphatic sulfonyl chlorides reacted efficiently with amines bearing sterically hindered amino group while the corresponding fluorides showed low activity.

Painting proteins blue: β-(1-azulenyl)-L-alanine as a probe for studying protein–protein interactions

We demonstrated that β-(1-azulenyl)-L-alanine, a fluorescent pseudoisosteric analog of tryptophan, exhibits weak environmental dependence and thus allows for using weak intrinsic quenchers, such as methionines, to monitor protein-protein interactions while not perturbing them.

A One-Pot Parallel Reductive Amination of Aldehydes with Heteroaromatic Amines

A parallel reductive amination of heteroaromatic amines has been performed using a combination of ZnCl2-TMSOAc (activating agents) and NaBH(OAc)3 (reducing agent). A library of diverse secondary amines was easily prepared on a 50-300 mg scale.