NMR Structure and Biophysical Characterization of Thermophilic Single-Stranded DNA Binding Protein from Sulfolobus Solfataricus

Abstract

Proteins from Sulfolobus solfataricus (S. solfataricus), an extremophile, are active even at high temperatures. The single-stranded DNA (ssDNA) binding protein of S. solfataricus (SsoSSB) is overexpressed to protect ssDNA during DNA metabolism. Although SsoSSB has the potential to be applied in various areas, its structural and ssDNA binding properties at high temperatures have not been studied. We present the solution structure, backbone dynamics, and ssDNA binding properties of SsoSSB at 50 °C. The overall structure is consistent with the structures previously studied at room temperature. However, the loop between the first two β sheets, which is flexible and is expected to undergo conformational change upon ssDNA binding, shows a difference from the ssDNA bound structure. The ssDNA binding ability was maintained at high temperature, but different interactions were observed depending on the temperature. Backbone dynamics at high temperature showed that the rigidity of the structured region was well maintained. The investigation of an N-terminal deletion mutant revealed that it is important for maintaining thermostability, structure, and ssDNA binding ability. The structural and dynamic properties of SsoSSB observed at high temperature can provide information on the behavior of proteins in thermophiles at the molecular level and guide the development of new experimental techniques.

The role of structural dynamics in the thermal adaptation of hyperthermophilic enzymes

“…Understanding the details of such optimisation is a crucial challenge in the quest of clarifying the general structure-function-activity relationship in protein molecules. Some studies have employed experimental techniques to probe protein dynamics at high temperature in fast ( Miletti et al, 2015 ; Yang et al, 2022 ) or slow ( Zavodszky et al, 1998 ; Butterwick and Palmer, 2006 ) dynamical regimes. MD simulations have also been extensively exploited in this research area ( Stafford et al, 2013 ; Katava et al, 2016 ; Stirnemann and Sterpone, 2017 ; Maffucci et al, 2020 ), however, it might be argued whether the simplifications in the empirical force fields represent critical limitations to elucidate the fine-tuning of protein dynamics at different temperatures.…”