Expression, Purification, and Assembly of Archaellum Subcomplexes of Sulfolobus acidocaldarius

Abstract

The archaellum assembly machinery and its filament consist of seven proteins in the crenarchaeon Sulfolobus acidocaldarius. We have so far expressed, purified, and biochemically characterized four of these archaellum subunits, namely, FlaX, FlaH, FlaI, and FlaF. FlaX, FlaH, and FlaI tightly interact and form the archaellum motor complex important for archaellum assembly and rotation. We have previously shown that FlaH forms an inner ring within a very stable FlaX ring, and therefore FlaX is believed to provide the scaffold for the assembly of the archaellum motor complex. Here we describe how to express and purify FlaX and FlaH and how the double ring structure both form can be obtained.

Motor torque measurement of Halobacterium salinarum archaellar suggests a general model for ATP-driven rotary motors

“…Motile archaea swim using surface appendages called archaella (previously named “archaeal flagella”), which are thrust-generating rotating helical filaments analogous to bacterial flagella 1 . Although the bacterial flagellum consists of >30 different proteins, the archaellum is built from as few as 7 proteins, none of which are homologous to flagellar proteins 2 . Instead, the archaellum is evolutionarily related to bacterial type IV pili.…”

Evidence for a compact σ70 conformation in vitro and in vivo

“…Next, to terminate the reaction, ammonium bicarbonate is added at three times the concentration of the crosslinker from a 1 M stock solution (so the sample is not diluted), and incubated at 25°C for 30 minutes with shaking. Later, preparation of the samples for mass spectrometry and mass spectrometry RAW data files analysis, is performed as previously described 78 , 79 with minor adjustments, see below the section on crosslinks identification and estimation of false detection rate ( FDR ).…”

“…Then samples are prepared for mass spectroscopy as previously described. 78 , 79 Briefly, the protein is precipitated in 1 mL of acetone (-80°C) for 1 hour, followed by centrifugation at 14,000 g. The pellet is resuspended in 20 mL of 8 M urea. The urea is diluted by adding 200 mL of digestion buffer (25 mM TRIS, pH = 8.0; 10% Acetonitrile).…”

Insights into the mechanism of ATP-driven rotary motors from direct torque measurement

“…To avoid additional confusion among another similar organelle in yet another different domain, archaeal flagellum was proposed to be denoted as "archaellum" (Jarrell and Albers 2012). The archaellum is built from as few as seven proteins with recent structural and functional studies providing a model of archaellar architecture Jarrell 2015, 2018;Chaudhury et al 2018;Jarrell and Albers 2012). To probe the mechanism of archaellar motility, the research team (including the current authors) have applied microscope techniques originally developed for single-molecule biophysics to the model swimming archaeon, Halobacterium salinarum (Hbt.…”