High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody.

Enhancing cytochrome P450-mediated non-natural cyclopropanation by mutation of a conserved second-shell residue.

Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non-native metallocarbenoid and -nitrenoid insertion reactions. P450-mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first-shell heme-ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450 .

P450-Mediated Non-natural Cyclopropanation of Dehydroalanine-Containing Thiopeptides.

Thiopeptides are a growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Many biosynthetic enzymes for RiPPs, especially thiopeptides, are promiscuous and can accept a wide range of peptide substrates with different amino acid sequences; thus, these enzymes have been used as tools to generate new natural product derivatives. Here, we explore an alternative route to molecular complexity by engineering thiopeptide tailoring enzymes to do new or non-native chemistry.

Non-natural carbenoid and nitrenoid insertion reactions catalyzed by heme proteins.

Despite increasing interest in using enzymes as tools for synthesis, many reactions discovered through the creativity of synthetic chemists remain beyond the scope of biocatalysis. This vacancy in the field has compelled researchers to develop strategies to adapt protein scaffolds for new reactivity. Heme proteins have recently been shown to activate synthetic precursors to generate reactive metallocarbenoid and metallonitrenoid species that enable the biosynthetic construction of novel C-C, C-N, and other bonds using mechanisms not previously explored by Nature.

An Evolved Orthogonal Enzyme/Cofactor Pair.

We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor.

Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation.

Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3 ) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst.