Maintenance of Native-like Protein Dynamics May Not Be Required for Engineering Functional Proteins

Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 A) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins

Design of a glutamine substrate tag enabling protein labelling mediated by <i>Bacillus subtilis</i> transglutaminase

<div><p>Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from <i>Bacillus subtilis</i> (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences were identified and kinetically characterized, with the most reactive sequence (YAHQAHY) having a k_cat/K_M value of 19 ± 3 μM^-1 min^-1. This sequence was then genetically appended onto a test protein as a reactive ‘Q-tag’ and fluorescently labelled with dansyl-cadaverine, in the first demonstration of protein labelling mediated by bTG.</p></div

Initial rates of bTG-mediated transamidation of POI-Q and dansyl cadaverine.

<p>The reaction of 0.8 μM of Q-tagged mRuby2 test protein with 5 mM dansyl cadaverine was mediated with 0.1 U of bTG over 20 min and detected using the GDH-coupled assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197956#sec010" target="_blank">Materials and methods</a>).</p

TGase-mediated protein cross-linking.

<p>Transamidation between protein-bound Gln and Lys residues leads to the formation of γ-glutamyl-ε-lysyl isopeptide bonds (red).</p

bTG-mediated fluorescent labelling of mRuby2 bearing high-affinity Q-tags.

<p>Purified test proteins bearing bTG recognition tags were fluorescently labelled with dansyl-cadaverine through bTG-mediated transamidation. SDS-PAGE gels of test proteins were run, followed by irradiation to visualize any fluorescent bands. After fluorescent visualization, Coomassie staining was performed to confirm the presence of protein bands.</p

FRET-based peptide screening assay.

<p>Cartoon representation of the conjugation of mRuby2-Q-tag and Clover-K-tag, in the presence of bTG, resulting in a cross-linked product. Due to the spectral overlap of mRuby2 and Clover, when in close proximity, excitation of Clover leads to FRET, and red emission by mRuby2.</p

Flow cytometry analysis of cells expressing peptide library.

<p>FACS plots of BL21(DE3) Gold cells expressing mRuby2-6NMT-Q plus Clover-6K, with and without bTG, at time points corresponding to 210 min (A, D), 240 min (B, E) and 300 min (C, F) after bTG induction. Plots are gated for putative FRET cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197956#pone.0197956.s001" target="_blank">S1 File</a>), excitation at 561 nm, emission at 614 nm (red FP channel) vs. excitation at 488 nm, emission at 620 nm (FRET channel). Plots A-C are of cells that do not contain bTG; plots D-F are of cells in which bTG expression has been induced. Gated cells and numbers of sorting events are colour-coded to identify distributions of populations from plot to plot (green = putative FRET, red = FRET positive, blue = background red fluorescence).</p

Protein logo of peptide library sequencing results.

<p>Protein logo representing the sequencing results for 78 sequences from the FRET-positive fraction of the 300-min mRuby2-6NMT-Q library. (<a href="http://rth.dk/resources/plogo/" target="_blank">http://rth.dk/resources/plogo/</a>).</p

Specificity constants of TGase-mediated transamidation.

<p>0.2–0.8 μM mRuby2-Qtag test proteins and 5 mM Gly-OMe were reacted in the presence of 0.1 U TGase. Initial rates were measured over 20 min, using the GDH-coupled assay. Specificity constants were determined by fitting initial rate data to the Michaelis-Menten equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197956#sec010" target="_blank">Materials and methods</a>).</p