Micro ATR FTIR imaging of hanging drop protein crystallisation

Abstract

Protein crystallisation is of great importance within structural proteomic projects where considerable time and effort is spent trying to obtain high resolution structural information from the X-ray diffraction of protein crystals. The crystallisation process is largely empirical due to a lack of understanding of crystallisation kinetics and it is often very challenging to obtain crystals of sufficient size and quality for X-ray analysis and data collection. This paper presents the use of micro Attenuated Total Reflection (ATR) -Fourier Transform Infrared (FTIR) spectroscopic imaging as an analytical method for the measurement of the growth of protein crystals using the hanging drop approach. As well as allowing for the measurement of in-situ growth of protein crystals, the high spatial resolution of this method allows the detection of micro-crystals. The use of this technique could be very advantageous for protein structural studies since it allows both detection of the growth of small protein crystals and provides chemical information as the crystals form.

Recent applications of ATR FTIR spectroscopy and imaging to proteins

“…This can be seen in the schematic of Figure 10. By measuring the growth of proteins in-situ, it is possible to detect the early stages of crystals grown and the formation of micro protein crystals, confirming the crystals are in fact protein rather than salt [87]. There may also by additional insight into the growth kinetic of protein crystals through analysis of secondary structure from the IR spectrum.…”

“…The high spatial resolution of micro ATR imaging means that it is particularly advantageous to the study of biological materials such as tissues, cells and arteries [72,86]. Most recently, this has been used to study hanging drop protein crystallisation [87]. A drop of protein and precipitant solution are deposited onto a removable Germanium ATR objective and suspended above a reservoir in exactly the same manner as conventional hanging drop crystallisation studies.…”

“…At only 64 μm x 64 μm [13], there is no way to guarantee that crystals will grow within this area. There could be potential to overcome this using seeding or surface modifications [87].…”

FT‐IR Imaging in ATR and Transmission Modes: Practical Considerations and Emerging Applications

“…This protocol was mimicked in a recent study in which the protein/precipitant solution was suspended from a Ge ATR crystal [99]. This protocol was mimicked in a recent study in which the protein/precipitant solution was suspended from a Ge ATR crystal [99].…”

“…This protocol was mimicked in a recent study in which the protein/precipitant solution was suspended from a Ge ATR crystal [99]. In situ micro-ATR FT-IR imaging of the protein crystallization (Reprinted from [99]. In situ micro-ATR FT-IR imaging of the protein crystallization (Reprinted from [99].…”