Sample Volume Reduction Using the Schwarzschild Objective for a Circular Dichroism Spectrophotometer and an Application to the Structural Analysis of Lysine-36 Trimethylated Histone H3 Protein.

Yudai Izumi,Koichi Matsuo

doi:10.3390/molecules23112865

Abstract

With the increasing interest in scarce proteins, reducing the sample volume for circular dichroism (CD) spectroscopy has become desirable. Demagnification of the incident beam size is required to reduce the sample volume for CD spectroscopy detecting transmitted light passed through the sample. In this study, the beam size was demagnified using a focal mirror, and small-capacity sample cells were developed in an attempt to reduce the sample volume. The original beam size was 6 × 6 mm2; we successfully converged it to a size of 25 × 25 μm2 using the Schwarzschild objective (SO). The new sample cell and SO allowed the required sample volume to be reduced to 1/10 (15 → 1.5 μL), when using a 15 μm path length cell. By adopting a smaller sample cell, further sample reduction could be achieved. By using the SO system, the secondary structural contents of the lysine-36 trimethylated histone H3 protein were analyzed. The trimethylation induced the increment of helix structures and decrement of unordered structures. These structural alterations may play a role in regulating cellular function(s), such as DNA damage repair processes.

Highlights

Circular dichroism (CD) spectroscopy in the ultraviolet (UV) region is widely used for the secondary structural analysis of proteins in an aqueous solution
The structural information from CD spectra is limited compared with that from X-ray crystallography and nuclear magnetic resonance (NMR), both of which display three-dimensional structures with atomic-level resolutions, CD spectroscopy is a powerful tool because it can more provide structural information, including the structural dynamics, because of some notable advantages: (i) the required sample amount is smaller
At the CD12 beamline of SRS in the U.K., the CD spectrum of myoglobin differed between the first and the second spectra, i.e., denaturation was induced by radiation damage within only 10 min [10], even though the photon flux density of the CD12 beamline was only 1% that of the focal position of the Schwarzschild objective (SO) system used in this study. These results indicate that protein samples placed

Summary

Introduction

Circular dichroism (CD) spectroscopy in the ultraviolet (UV) region is widely used for the secondary structural analysis of proteins in an aqueous solution. The structural information from CD spectra is limited compared with that from X-ray crystallography and nuclear magnetic resonance (NMR), both of which display three-dimensional structures with atomic-level resolutions, CD spectroscopy is a powerful tool because it can more provide structural information, including the structural dynamics, because of some notable advantages: (i) the required sample amount is smaller (1–10% of those needed for X-ray crystallography and NMR [1]) and (ii) the samples can be prepared by dissolving the proteins in a solvent. The use of synchrotron radiation (SR) as a light source for CD spectroscopy allows more precise structural information to be obtained, because an SR beam can expand the measurement region to the vacuum-ultraviolet (VUV) region where additional CD peaks are often observed. Synchrotron radiation circular dichroism (SRCD) spectroscopy has produced successful outcomes over the past

Methods

Results

Conclusion