Backbone dynamics of TFE‐induced native‐like fold of region 4 of Escherichia coli RNA polymerase σ⁷⁰ subunit

Abstract

Folding of a recombinant protein rECsigma(70) (4) comprising domain 4 of E. coli RNA polymerase sigma(70) subunit, upon addition of 2,2,2-trifluoroethanol (TFE) to its aqueous solution, was monitored by heteronuclear NMR spectroscopy. The TFE-induced migration of resonance signals in a series of (15)N-HSQC spectra displayed sequence-dependent heterogeneity. A common trend of uniform upfield shift in both (1)H and (15)N dimensions, indicative of generation of helical structures, breaks down for some residues almost cooperatively at 10-15% TFE (v/v), pointing to the buildup of non-helical regions separating the initially induced helices. The preferences of residues to assume either helical or non-helical conformation are correlated with the location in the sequence rather than with their type. CSI descriptors and (15)N relaxation data obtained for the protein at 10% TFE allowed characterization of the stability of the pre-folded state of rECsigma(70) (4). By all the criteria applied, three highly populated alpha-helical regions separated by much more flexible residues forming a loop and a turn in the DNA-binding HLHTH motif were identified. The location of the secondary structure elements along the protein sequence coincides with those found in homologous proteins, and with the helix nucleation regions determined in unfolded rECsigma(70) (4) at low pH. The bimodal distribution of the (15)N relaxation parameters enabled identification of residues forming a framework of the folded protein strictly corresponding to the HLHTH motif, bracketed by unfolded terminal regions. Thus, in respect to rECsigma(70) (4) in aqueous solution TFE acts not only as a strong helix inducer, but also as a folding agent.

The TFE-induced transient native-like structure of the intrinsically disordered $$\varvec\sigma_{ 4}^{ 70}$$ σ 4 70 domain of Escherichia coli RNA polymerase

“…1 b). The average population of helical structures in region 4.2 reaches 0.6 for a 30 % (v/v) TFE solution, while at 10 % (v/v) TFE it approached only 0.4 (Kaczka et al 2010 ), respectively 0.33 and 0.37, when estimated using δ2d. The average population of helical structures for the whole protein was estimated to be 0.25 (Kaczka et al 2010 ) and 0.38 for 10 and 30 % TFE solutions and respectively 0.25 and 0.26 when calculated using the δ2D approach.…”

“…A consistent pattern of the downfield shifts of 13 Cα and 13 CO resonances, accompanying the upfield shift of the 1 Hα, was observed for numerous residues. The values of the score function, (see (Kaczka et al 2010 ) for details) identify significantly populated helical patterns, located in the regions: 3.2 (L532-A542; H0), 4.1 (L551-F563; H1) and 4.2 (L573-L599; H2, H3). Another less populated fragment, S602-S609, is located in the C-terminal part of (Fig.…”

“…The helical population of individual residues, p α ( i ), was estimated as the ratio of Δδ( i )/Δδ o ( i ), where the reference values of secondary chemical shifts, Δδ o ( i ), were assumed to be 2.8, 2.3 and −0.4 ppm for 13 Cα, 13 CO and 1 Hα, respectively (Wishart and Sykes 1994 ; Schwarzinger et al 2000 ). In view of the consistency of the Δδ( i ) distribution pattern for 13 Cα, 13 CO and 1 Hα, the consensus local propensities toward a helical structure were expressed as the geometric average of the three descriptors referred to above (Kaczka et al 2010 ). The population of helical structures were additionally estimated on the δ2D server (uk/d2D/ Jobs submitted: July 2014 ), the algorithm of which is based on the larger set of chemical shift data ( 1 Hα, 13 Cα, 13 Cβ, 13 CO, N and HN) (Camilloni et al 2012 ).…”

“…Since the studied protein was known to be highly unfolded (Poznanski et al 2003 ; Kaczka et al 2010 ), the standard Lipari-Szabo model-free approach (Lipari and Szabo 1982a , b ), which attributes a global correlation time ( τ m ) to the overall rotational diffusion, was found inappropriate (Alexandrescu and Shortle 1994 ; Brutscher et al 1997 ). Hence, an alternative approach of the direct fitting of the spectral density function (Eq.…”

“…This generally agrees with the idea that TFE induces the closure of individual hydrogen bonds (Jaravine et al 2001 ) and thus propagates the context-dependent formation of native-like secondary structures in polypeptides (Lawrence and Johnson 2002 ). Subsequently, we have monitored the TFE-induced conformational changes of with the aid of NMR spectroscopy (Kaczka et al 2010 ). The drift of resonance signals in the 1 H- 15 N HSQC spectra indicated that in a mixed water-TFE solution, a non-specific uniform build-up of helical structures takes place for a THE content smaller than 10 % (v/v).…”