High-throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

Abstract

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single-molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high-throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.

Figure 1

Assessment of protein aggregation using FCS. (a–d) ¹H-¹⁵N HSQC spectra of MIP-1α at various pH levels. (e) FCS measurement of MIP-1α in pH titration.

Figure 2

High-throughput screening of optimal solution conditions using FCS. (a) FCS measurements of CERT PH domain at various pH and NaCl concentrations. (b) FCS measurements of CERT PH domain with various chemical additives at pH 7.5. (c,d) ¹H-¹⁵N HSQC spectra of CERT PH domain.