Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Summary

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

Abstract

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding.

Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Introduction

Much of the human proteome remains poorly characterized. The "streetlight effect" described by Dunham and Kustatscher et al. refers to the phenomenon where extensively researched proteins receive more attention, leaving understudied proteins overlooked^1,2. Factors contributing to this effect include the biological importance and disease relevance of certain proteins. Moreover, prior research that offers foundational knowledge, as well as the availability of tools for functional analysis, further stimulates research on highly studied proteins^1,2....

Protocol

1. Experimental setup

1. Use a thermal cycler that can detect fluorescence, such as an RT-PCR instrument, to perform the described protocol. If using SYPRO Orange, as described in this protocol, use a scanning mode that allows for excitation around 470 nm and emission detection around 570 nm. Program the thermocycler to hold the sample at 20 °C for 2 min, followed by an increase in temperature of 0.5 °C/1 min up to 95 °C; measure fluorescence intensity every 1 °C.
   NOTE.......

Discussion

The Thermal Shift Assay (TSA) serves as an efficient method for screening protein-ligand interactions, including those with cofactors and inhibitors. In this protocol, we used TSA to measure the binding of the pseudokinase SelO to nucleotides and divalent cations. Our findings show that SelO exhibits increased thermal stability in the presence of ATP and Mg²⁺/Mn²⁺. This observation aligns with previous reports indicating that SelO homologs from E. coli, S. cerevisiae, and H. sapiens

Materials

Name	Company	Catalog Number	Comments
Adenosine 5′-triphosphate disodium salt hydrate	Sigma Aldrich	A2383-10G	used for representative results but not required to perfom TSA
Avanti J-15R with microplate carrier assembly	Beckman Coulter	C19416
CFX Opus 384 Real-time PCR system	Bio-Rad	12011452
Hard-Shell 384-Well PCR Plates, thin wall, skirted, clear/white	Bio-Rad	HSP3805
Magnesium Chloride	Sigma Aldrich	M8266-100G	used for representative results but not required to perfom TSA
Manganese (II) chloride tetrahydrate	Sigma Aldrich	M3634-500G	used for representative results but not required to perfom TSA
Microseal 'B' PCR Plate Sealing Film, adhesive, optical	Bio-Rad	MSB1001
SYPRO Orange Protein Gel stain	Sigma Aldrich	S5692-500UL
Uridine 5′-triphosphate trisodium salt hydrate	Sigma Aldrich	U6625-100MG	used for representative results but not required to perfom TSA