Perspective: A toolbox for protein structure determination in physiological environment through oriented, 2D ordered, site specific immobilization

Abstract

Revealing the structure of complex biological macromolecules, such as proteins, is an essential step for understanding the chemical mechanisms that determine the diversity of their functions. Synchrotron based X-ray crystallography and cryo-electron microscopy have made major contributions in determining thousands of protein structures even from micro-sized crystals. They suffer from some limitations that have not been overcome, such as radiation damage, the natural inability to crystallize a number of proteins, and experimental conditions for structure determination that are incompatible with the physiological environment. Today, the ultra-short and ultra-bright pulses of X-ray free-electron lasers have made attainable the dream to determine protein structures before radiation damage starts to destroy the samples. However, the signal-to-noise ratio remains a great challenge to obtain usable diffraction patterns from a single protein molecule. With the perspective to overcome these challenges, we describe here a new methodology that has the potential to overcome the signal-to-noise-ratio and protein crystallization limits. Using a multidisciplinary approach, we propose to create ordered, two dimensional protein arrays with defined orientation attached on a self-assembled-monolayer. We develop a literature-based flexible toolbox capable of assembling different kinds of proteins on a functionalized surface and consider using a graphene cover layer that will allow performing experiments with proteins in physiological conditions.

Immobilization of Proteins with Controlled Load and Orientation

“…With this expanded ability for precise construction of protein-based biomaterials, it will become possible to meaningfully define how protein function and stability are affected by the interplay between surface-dependent variables such as roughness, nanostructure, and electrostatics with standard protein-dependent variables such as quantity, orientation, and density. , For example, precise control over orientation will permit activity density to be maximized (e.g., Figure D) and thereby facilitate further miniaturization in protein-based biosensors. , Additionally, achieving simultaneous control over protein density and orientation in protein microarrays would simplify their construction and quality which could expand their use in proteomics. , Furthermore, such density-controlled, homogeneously oriented protein surfaces could provide the highly homogeneous samples that are required for atomic-level structural determination via techniques such as cryoelectron microscopy, solid-state nuclear magnetic resonance, , and coherent diffraction imaging, which, as of yet, has not been feasible. Finally, this approach has the potential to seamlessly integrate with other emerging surface technologies, such as nonfouling coatings and spatially organized nanostructures, that together promise to not only generate multifunctional biomaterials with enhanced performance but also further our understanding of the forces that impact protein structure and function on material surfaces.…”

“…5,46 Additionally, achieving simultaneous control over protein density and orientation in protein microarrays would simplify their construction and quality which could expand their use in proteomics. 47,48 Furthermore, such densitycontrolled, homogeneously oriented protein surfaces could provide the highly homogeneous samples that are required for atomic-level structural determination via techniques such as cryoelectron microscopy, 49 solid-state nuclear magnetic resonance, 50,51 and coherent diffraction imaging, 52 which, as of yet, has not been feasible. Finally, this approach has the potential to seamlessly integrate with other emerging surface technologies, such as nonfouling coatings 53 and spatially organized nanostructures, 54 that together promise to not only generate multifunctional biomaterials with enhanced performance but also further our understanding of the forces that impact protein structure and function on material surfaces.…”