Controlling complexity and water penetration in functional de novo protein design

Abstract

Natural proteins are complex, and the engineering elements that support function and catalysis are obscure. Simplified synthetic protein scaffolds offer a means to avoid such complexity, learn the underlying principles behind the assembly of function and render the modular assembly of enzymatic function a tangible reality. A key feature of such protein design is the control and exclusion of water access to the protein core to provide the low-dielectric environment that enables enzymatic function. Recent successes in de novo protein design have illustrated how such control can be incorporated into the design process and have paved the way for the synthesis of nascent enzymatic activity in these systems.

Elementary tetrahelical protein design for diverse oxidoreductase functions

“…The formation and lifetime of an oxyferrous heme state provides a sensitive probe of heme-ligand affinities, exchange dynamics and water accessibility to the heme site 13 . Crucial for successful oxygen binding is the introduction of strain into the helices during the modeled mechanical rotation upon bis -histidine ligation of the heme 14 .…”

“…This includes light- and redox-active cofactors supporting oxidation and reduction 10 , proton coupling 11 , electrochemical charge coupling 12 and ligand exchange 13 , including the generation of a stable oxyferrous heme state familiar in oxygen transport by globins 14 . However, the sequence duplication of these symmetrical homotetrameric and homodimeric structures fails to support the diverse, multiple cofactor assembly needed for more sophisticated oxidoreductases.…”

Maquette Strategy for Creation of Light- and Redox-Active Proteins

“…For example, the rotational strain on the histidine ligations to the heme iron promoted by locally acting interfacial glutamates in helical column [Koder et al, 2009] continued to support the formation, dynamics and stability of the oxyferrous state. Similarly, the disulfide link to the homodimer essential for preventing water access and loss of the oxyferrous heme [Anderson et al, 2008] was functionally replaced by the diagonal loop linkage in the single-chain structure. In maquette redesign, transfers between earlier structural forms to the single-chain four-a-helix structure are manageable and quantifiable.…”

“…With the loop linkage in place, an oxyferrous state was stably formed on the same timescale with oxygen affinities and exchange timescales matching those of the natural globin family. It has become clear that this simple link introduced to constrain inter-helical dynamics does not impede access of small molecules like dioxygen and carbon monoxide into the maquette interior but raises a barrier that slows the access of water by orders of magnitude to suppress electron transfer with the loss of the oxyferrous bond to produce ferric heme and superoxide [Anderson et al, 2008]. As was concluded by Koder et al [2009], "… the ease with which globin-like properties can be reproduced in a completely unrelated and simply engineered maquette indicates that the relatively complex globin fold is for the most part unremarkable, and may be common in nature not because of a uniquely capable design for oxygen binding, but simply because it is good enough.…”

Bioinspired approach toward molecular electrets: synthetic proteome for materials

“…At systems level, the implementation of biomimesis allows for testing the hierarchy of importance of the various structure-function relationships in the natural systems. In the process of understanding the molecular and cellular aspects of living organisms, numerous examples of artificial enzymes [41][42][43][44], synthetic de novo designs of proteins [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59], artificial photo-driven proton pumps [60,61], and artificial cells with designated functionalities [62][63][64][65] have advanced the fields of biology, biochemistry and biophysics and provided unprecedented paradigms for bioengineering. At an organism-size level, examples for walking machines, which do not utilize wheels or roller, may seem somewhat impractical for the "optimal practical usage" of the current state of robotics.…”