Alan J. Kennan

A synthetic peptide ligase

The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 104. These results augur well for the rational design of functional peptides.

Orthogonal Recognition in Dimeric Coiled Coils via Buried Polar-Group Modulation

We describe the design and exploration of new buried polar groups to control coiled-coil dimerization. Employing our recently described method for on-resin guanidinylation, we have prepared coiled-coil peptides with a single core guanidine, spaced from the backbone by 1-3 methylene groups. Heterodimeric mixtures of these sequences with guanidine, amide, and carboxylic acid binding partners form a large number of reasonably stable coiled coils (T(m) > or = 60 degrees C). A detailed stability trend examination reveals that asparagine/acid pairs are sharply sensitive to acid residue chain length (Asn/Asp much worse than Asn/Glu), while guanidine/acid pairs are largely insensitive. This has been exploited to create orthogonal recognition pairs which establish the capacity to form two distinct heterodimeric coiled coils by simple mixing of four different peptides. One dimer has buried core asparagines, while the other pairs aspartic acid with any of three guanidinylated side chains. Specificity of this behavior is underscored by failure of glutamic acid substituted sequences to perform accordingly. The successful alternate pairs are further characterized by various biophysical methods (circular dichroism, ultracentrifugation, thermal and chemical denaturation, affinity tags).

Simultaneous directed assembly of three distinct heterodimeric coiled coils.

We describe simultaneous formation of three distinct heterodimeric coiled coils from a mixture of six different peptides. The choice among electrostatically viable complexes is governed by alignment of buried core residues, including a fundamentally new interaction that exploits urea-terminated side chains. Buried urea/urea contacts lead to extremely stable dimeric coiled coils, with T(m) values between 63 and 79 degrees C. Core ureas can also form stable complexes with a variety of other polar groups, including guanidines, acids, and amides.

GLUE that sticks to HIV: a helix-grafted GLUE protein that selectively binds the HIV gp41 N-terminal helical region.

Methods for the stabilization of well‐defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Here, we report the stable and functional display of an HIV gp41 C‐peptide helix mimic on a GRAM‐Like Ubiquitin‐binding in EAP45 (GLUE) protein. C‐peptide helix‐grafted GLUE selectively binds a mimic of the N‐terminal helical region of gp41, a well‐established HIV drug target, in a complex cellular environment. Additionally, the helix‐grafted GLUE is folded in solution, stable in human serum, and soluble in aqueous solutions, and thus overcomes challenges faced by a multitude of peptide drugs, including those derived from HIV gp41 C‐peptide.

Heterotrimeric Coiled Coils with Core Residue Urea Side Chains

We report several coiled coil heterotrimers with varying core residue buried polar groups, all with T(m) values >43 degrees C. Introduction of new synthetic side chain structures, including some terminating in monosubstituted ureas, diversifies the pool of viable core residue candidates. A study of core charge pairings demonstrates that, unlike dimeric systems, trimeric coiled coils do not tolerate guanidine-guanidine contacts, even in the presence of a compensating carboxylate. Overall, the roster of feasible coiled coil designs is significantly expanded.

Protein structure: Charting a new course in coiled coils.

Designed expansion of hydrophobic contacts converts a coiled-coil tetramer to a true hexamer, a new protein fold previously deemed unlikely to exist. The complex has a central channel sized to allow passage of water molecules.

Helix-Grafted Pleckstrin Homology Domains Suppress HIV-1 Infection of CD4-Positive Cells.

The size, functional group diversity and three‐dimensional structure of proteins often allow these biomolecules to bind disease‐relevant structures that challenge or evade small‐molecule discovery. Additionally, folded proteins are often much more stable in biologically relevant environments compared to their peptide counterparts. We recently showed that helix‐grafted display—extensive resurfacing and elongation of an existing solvent‐exposed helix in a pleckstrin homology (PH) domain—led to a new protein that binds a surrogate of HIV‐1 gp41, a validated target for inhibition of HIV‐1 entry. Expanding on this work, we prepared a number of human‐derived helix‐grafted‐display PH domains of varied helix length and measured properties relevant to therapeutic and basic research applications. In particular, we showed that some of these new reagents expressed well as recombinant proteins in Escherichia coli, were relatively stable in human serum, bound a mimic of pre‐fusogenic HIV‐1 gp41 in vitro and in complex biological environments, and significantly lowered the incidence of HIV‐1 infection of CD4‐positive cells.