Lanthanide Binding Tags as Self-Assembling Gd(III) Spin-Labels for In Vivo and In vitro Nanometric Distance Determination in Proteins

RODOLFO M. RASIA; SUN UN; H. Y. VINCENT CHING; LEANDRO C. TABARES; FLORENCIA C. MASCALI; ROBERTA CRESPO

Nansen

Congreso; EUROMAR 2018; 2018

Nanometric distances determination by Pulse Electron Double Resonance (PELDOR or DEER) has become an important tool in structural biology. PELDOR measures the distance between two paramagnetic centers by determining the strength of theirmagnetic interaction. These paramagnetic centers are in general introduced in vitro by chemical reaction of cysteines at the protein surface with sulfur-reactive spin-labels such derivatives of nitroxide radicals or DOTA chelator. Such a labeling approach requires unique cysteines and cannot be accomplished inside cells. A different approach is to genetically encode a peptide sequence capable of coordinating a paramagnetic metal ion that can subsequently form self-assembled metal spin-labels in vivo. Mn(II) bound to His-Tags and Gd(III) bound to Lanthanide Binding Tags (LBT) are two examples of such self-assembled metal spin-labels (SAMSL) that have been used for in vitro PELDOR measurement. We have studied whether these SAMSL can be biosynthetically produced inside the cell and directly used for in-cell distance measurements. To this end, two LBTs were genetically fused to the ends of a small 3-helixbundle(3Hx) protein and the constructs expressed in E. coli. A clear PELDOR response arising from Gd(III):LBT-3Hx-LBT:Gd(III) synthesized by the cells was detected on the purified protein. More importantly, the same signal was detected directly from the cells overexpressing the protein grown on media supplemented with Gd(III).In addition to the in vivo studies, LBTs are also helpful for studying cysteines-rich proteins. HYL1 is a protein involved in the miRNA processing on A. thaliana. This protein has two independent domains connected by a flexible linker. The independent structure of each domains is known; however, structural information of the HYL1/RNA complex is yet unknown. HYL1 contains 5cysteines and it has proven difficult to express HYL1 mutants carrying only two exposed cysteines residues due to poor protein stability. Instead, we have decided to use Gd(III):LBT SAMLS-tags. Three constructs were prepared, each of them carry two LBTs: N-Ter/C-Ter (HYL1-L12L), N-Ter/central loop (HYL1-L1L2) and central-loop/C-Term (HYL1-1L2L). PELDOR measurements were carried out on these constructs and their complexes with RNA.The results on these systems as well as the advantages/disadvantages and possible improvements for the use of LBT-SAMLS in in vivo and in vitro nanometric distance determination will be discussed.