Cotranslational protein folding on the ribosome monitored in real time

Abstract

The gene coding for the protein methyl transferase HemK (ECBD_2409, 834 bp, 277 aa) was amplified from the genomic DNA of E. coli BL21 DE3 by colony PCR using the primers 5'-GTCCGAGCAGGACATATGGAATATCAA-3' and 5'-GCAGTGTAG AAAAACCTCGAGTTGATAAT-3', digested with NdeI and XhoI (New England Biolabs) and ligated into a pET-24a Vector (Novagen). The gene sequence encoding for the N-terminal domain of E. coli HemK (residues 1-73, HemK NTD hereafter) was amplified by PCR from a vector encoding full-length HemK (residues 1-277), using primers containing recognition sequences for NdeI and XhoI restriction enzymes for subsequent cloning into expression vector pET24-a (Novagen). The purified PCR fragment was digested with NdeI and XhoI restriction enzymes for 1 h and ligated into the expression vector pET-24a linearized with the same restriction enzymes. The Leu-Glu sequence (encoded by the XhoI restriction used for cloning) was subsequently removed by inverse deletion PCR. Mutants of wild type NTDHemK were made by PCR using a commercial site-directed mutagenesis kit (Stratagene, Carlsbad, CA) according to the manufacturer's instructions. We note that HemK from E. coli BL21 DE3 contains Lys at position 34, in contrast to HemK from E. coli K12, which contains Arg at position 34 and the structure of which is available. In the following, the sequence ofHemK from E. coli BL21 DE3 is denoted as wild type (wt). Endogenous tryptophan residues at position 6 and 78 were replaced with phenylalanine in all constructs using the quick-change method. For PET experiments tryptophan residues were introduced at position 6, 38, and 49 using site-directed mutagenesis. All constructs were verified by sequencing. Expression and purification of HemK NTDThe plasmid encoding HemK NTD (or its mutants) was transformed into competent E. coli Fluorescence emission spectra and equilibrium chemical unfoldingFluorescence emission spectra were recorded at 25 °C with a Horiba model Fluorolog 3 spectrofluorimeter connected to a circulating water bath. Measurements were performed in a quartz cuvette of 1-cm path length at a protein concentration of 2 µM in buffer E.Fluorescence was excited at 280 nm. Fluorescence emission spectra were collected between 310 and 420 nm in 1 nm increments and were corrected for background (buffer without protein). Slid widths of 1 and 10 nm were used for excitation and emission, respectively. Samples were incubated for 2 hours before measurements were taken. For each collected background-corrected fluorescence emission spectrum, the normalized global emission

The ribosome regulates flavodoxin folding

“…As explained above, the circumstances under which proteins are introduced in in vivo or in vitro experiments (i.e., full-length sequence versus growing polypeptide) differ considerably. Though in some cases chemically or sequentially truncated versions of proteins have been studied for their folding behaviour (21)(22)(23)(24), these experiments do not take into account the other major differences between in vivo and in vitro circumstances. As the lifetimes of proteins in cells can range from minutes to years (25) there is ample time for multiple folding rounds for fully synthesized protein.…”

“…The construct used for RNC production consists of several components ( Fig. 5) (38,57): a triple Strep-tag for purification purpose; a Smt3-domain with a C-terminal Ulp1-protease cleavage site to produce authentic N-termini for RNCs; the flavodoxin sequence; a TEV-protease site located C-terminally of the flavodoxin sequence to enable release of the nascent chain from the ribosome; a linker that spans the ribosomal exit tunnel; and the SecM sequence, which stalls the nascent chain to the ribosome.…”

“…In contrast, off-pathway MGs contain non-native secondary structure and/or packing. Nowadays, many proteins are thought to fold via MGs (17)(18)(19)(20)(21)(22)(23) and these intermediates are for example necessary during insertion of proteins into membranes or during translocation (24)(25)(26). Because of their importance in protein folding, considerable effort has been put into determining the structural properties of MGs.…”

“…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:…”

“…Most cofactors bind non-covalently, and in case of flavoproteins ninety percent bind flavin in this way (24). Flavoproteins make up to three and a half percent of genes in certain genomes and fulfil many important roles (24).…”