Nanosecond Dynamics of Calmodulin and Ribosome‐Bound Nascent Chains Studied by Time‐Resolved Fluorescence Anisotropy

Abstract

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

“…Some proteins seem to fold only when they are entirely exposed outside the ribosome [12] or when they are released [18]. During synthesis, other proteins can form folding intermediates [8,9,11,13,17,19], and protein subdomains may properly fold while the remainder of the nascent sequence does not [14,16]. In addition, the ribosome itself can in principle also modulate nascent chain folding [20].…”

“…Folding can already begin co-translationally, with only part of the polypeptide chain synthesized, and has been investigated for protein folds like all α [7][8][9], all β [10][11][12][13] and α/β [14][15][16][17]. Some proteins seem to fold only when they are entirely exposed outside the ribosome [12] or when they are released [18].…”

Nonorthogonal tRNAcysAmber for protein and nascent chain labeling

“…Unfortunately, the presence of the ribosome hampers standard labeling of RNCs, necessitating the use of alternative strategies. With the exception of a recently applied biarsenical fluorescein derivative (FlAsH) (Lamprou et al 2014), fluorophores are introduced into RNCs during translation through the use of either (i) fluorescent unnatural amino acids (Saraogi et al 2011) or (ii) tRNA charged with a standard amino acid, which is subsequently modified with a fluorophore (Flanagan et al 2003;Woolhead et al 2004;Ellis et al 2008;Lin et al 2012).…”

The ribosome regulates flavodoxin folding

“…Decay of fluorescence anisotropy of apoflavodoxin is caused by exchange of excited-state energy between two tryptophan residues by the Förster mechanism and by overall protein rotation and is described by (37,56,57): Reversible energy transfer happens between a pair of identical, isoenergetic chromophores (i.e., tryptophans). In this case φ T is given by:…”

“…Such insertions often happen cotranslationally, i.e., while the ribosome synthesizes the polypeptide concerned. Proteins can fold cotranslationally and sample intermediate folding states (51)(52)(53)(54)(55)(56)(57), which might include MGs.…”