[[The]] characterization of flow-induced protein crystallization at the air/water interface

Abstract

Proteins are an essential part of any organism as they are involved in nearly all known biological functions. The specific function of a protein is heavily dependent on its three-dimensional structure at the molecular level. Many advances in biology and medicine have been made possible by the detailed structural study of proteins. Furthermore, developing a better understanding of protein structure could lead to purposefully designed macromolecules. The most widely used and powerful technique to determine the structure of a protein is X-ray crystallography. However, in order for this method to be used, the protein must first be crystallized. Crystallizing proteins is usually not trivial and the success rate is often dismal. One approach is two-dimensional crystallization at the air/water interface. This entails the specific binding of protein initially in solution to a ligand that has been previously spread on the interface to form an insoluble monolayer.

Common ways to determine if a protein has crystallized is to use fluorescently tagged proteins or to lift the surface film onto a solid substrate, such as mica, and perform microscopy on the lifted sample. The results of this study demonstrate that the surface shear viscosity provides a sensitive macroscopic probe to detect flow-induced two-dimensional crystallization. It is shown that the formation of crystals corresponds to an abrupt change in the surface shear viscosity, providing an <italic>in situ</italic> indicator of crystal formation, and allowing for the real-time non-invasive analysis of the crystallization process with a macroscopic tool. Furthermore, by performing experiments at a wide range of conditions, results are presented that delineate the shear rate that is required to induce the formation of two-dimensional protein crystals for a given surface pressure. Finally, analogies are drawn from the phenomenon of flow-induced crystallization of bulk polymer melts to propose a possible qualitative thermodynamic explanation for the propensity of a protein crystal to be induced by flow.

In practice, two-dimensional protein crystallization has been done in quiescent systems with various degrees of success. Recently, it was shown that a bulk flow can enhance two-dimensional protein crystallization by inducing crystallization at a surface pressure where the protein cannot crystallize in a quiescent system. In the present research, the fundamental physics for flow-induced crystallization is examined. This study involves the use of the deep-channel surface viscometer, which consists of an annular flow regime bound by stationary inner and outer cylinders and is driven by a constant rotation of the floor. This geometry allows for the direct measurement of the surface shear viscosity as an indication of the meso-scale interactions that occur during the crystallization process. The results of this study indicate that interfacial strain rate is responsible for flow-induced protein crystallization in two dimensions, similar to flow-induced crystallization in polymer melts in three dimensions.