De novo design of catalytic and membrane proteins

There are four research areas in the Department of Pharmaceutical Chemistry. De novo design of catalytic and membrane proteins is a research challenge within protein and cellular engineering.

The challenge

Determining how the initial linear sequence of amino acids (residues) that comprise a protein dictates the specific, complex structure that it folds into. This includes discovering the underlying principles that lead proteins comprised of almost entirely different residues to fold into similar structures.

While structural analysis and site-directed mutagenesis offer critical insights into the principles underlying protein folding and how physicochemical details determine biological activity (structure-activity relationships), the de novo design of simplified but functional proteins puts the understanding of those principles to complementary and critical tests.

Ultimately, this engineering should allow researchers to better understand the protein malformations that can underlie some disease as well as how to design and make structurally novel proteins that function as therapies.

Examples of our research and methods include

Designing metallo-proteins, membrane transporters

computationally designed transmembrane protein (PRIME)

Ivan V. Korendovych, PhD

A computationally (de novo) designed transmembrane protein that binds two non-natural iron diphenylporphyrins. Dubbed PRIME (for PoRphyrins In MEmbrane), its four-helix bundles and metallo-co-factors form a path for electron transfer across the membrane.

Having pioneered in de novo design of water-soluble alpha-helical bundles and synthetic membrane-spanning ion channels, department scientists have more recently computationally designed and genetically expressed self-assembling functional models of metalloproteins—both water soluble and transmembrane—that selectively bind specific hemes, unique non-natural porphyrins, and di-metal ion cofactors.

These designed proteins have included functional models of cytochromes and other proteins that transfer electrons across cellular membranes—a process that is, for example, key to the production of ATP, the molecule that transports energy to fuel the activities of life in our cells.

The design of model co-transporters from first principles is also ongoing. These transmembrane proteins use the gradient of one ion to drive the transport of another; for example, concentrating nutrients in a cell or removing toxins.

The design of model metalloproteins helps address key questions about how their abundant natural counterparts—up to a third of proteins employ metal in function— draw on a limited palette of amino acids and metal ions to produce a great diversity of catalytic functions. In particular, minimal scaffolds allow for focus on how changes to the active site yield that variety, imposing a specific geometry on bound cofactors and tuning their properties.

Likewise, the challenging engineering of transmembrane proteins, which play critical roles as channels, transporters, receptors, and thus pharmaceutical targets, tests emergent principles regarding the folding of proteins translocated to cell membranes. In the latter environment, hydrophobic forces in the cytosol give way to the assembly of linked subdomains via polar interactions and shape complementarity.

Such model proteins are also easier to systematically alter, incorporating non-natural cofactors, and to study, via spectroscopic probes, isotopic labels, and elements for surface immobilization.

Our applications of such protein engineering include

Reprogramming a protein via remodeling

Rationally reprogramming a de novo designed di-iron carboxylate protein by remodeling the substrate access cavity and adding a third histidine ligand to the metal binding cavity. The structural alterations changed the protein’s substrate (hydroquinones to arylamines), reaction type (two-electron oxidation to N-hydroxylation), and relative reaction rate (at least a million-fold increase).

a: Surface models of a de novo designed 114-residue four-helix bundle di-iron binding Due Ferri protein (DFsc, top) and the redesigned carboxylate enzyme (G4DFsc) with four glycine residues substituted for alanines (shown in white), opening up the substrate access channel.
b: Another variant (3His-G2DFsc) with a third metal-binding histidine residue added to the active site (H100) with supporting mutations in the interior of the four-helix bundle. (Viewer-facing helix is translucent).

These simple changes reprogram the reactivity of the resulting protein from hydroquinone oxidation to selective arylamine N-hydroxylation.

Synthesizing electron-transferring membrane proteins

Synthesizing a four-helix transmembrane metalloprotein complex that assembles without the hydrophobic driving force and binds two non-natural co-factors with high affinity and specificity. Dubbed PRIME (PoRphyrins In MEmbrane), the membrane protein positions two iron diphyenylporphyrins close enough to provide a pathway for electron transfer across the membrane bilayer.

Synthesis of the four-helix porphyrin-binding bundles:

a: hydrophilic residues show in blue, hydrophobic residues in green, and metal cofactor in brown
b: Design of the iron coordination site in PRIME
c: General approach to PRIME design
d: Final repacked model of PRIME inserted in bilayer (yellow).