Circular permutation of LucY.

<p>(a) Domains 1 and 3 were connected with a small linker (dashed line), and new N and C-termini were created by making Break Points (BP) between domains 1 and 2 (BPs1-5) or between domains 2 and 3 (BPs6-10). (b) Fluorescence of circularly permutated LucY molecules was compared to wild type. (c) Split points were made from a circular permuted LucY such that domains 3 and 1 make up one half and domain 2 makes up the other. Fluorescence of the co-expressed pair is represented as a percentage of CZ. Image above each graph shows whole cell pellets of corresponding sample under UV light. Error bars represent standard deviation from the mean (n = 3).</p

Crystal structure of LucY.

<p>(a) LucY crystal trials as seen under UV light (left and middle panel) and visible light (right panel). (b) Left, three-domain fold of LucY. Domains 1 (blue), 2 (white), 3 (pink), and linker loops (yellow) are shown. Right, structure alignment of MurB enzymes: LucY (green), S. aureus (cyan), E. coli (white), and T. caldophilus (yellow). LucY is a type I MurB which lacks the βαββ fold and Tyr 190 loop found in E. coli. FAD cofactors are shown as sticks, nitrogen in blue, oxygen red, and phosphorus orange.</p

Excitation/emission of LucY.

<p>Excitation scan (black) performed with emission at 528nm and emission scan (gray) was performed with excitation at 465nm.</p

LucY reassembly.

<p>(a) Five split points between domains 1 and 2, referred to as SPa NZ or CZ 1–5, were fused with leucine zippers and co-expressed. Image above each graph shows whole cell pellets of corresponding sample under UV light. Fluorescence is represented as a percentage of CZ. Error bars represent standard deviation from the mean (n = 3). (b) Five split points between domains 2 and 3 (SPb NZ or CZ 1–5) were fused with leucine zippers and co-expressed. (c) SPbNZ5 was paired with other split points to determine pair with highest fluorescence. (d) SPbNZ5 paired with either SPbCZ1 or SPbCZ5, was tested for fluorescence independently and without leucine zipper (ΔZip).</p

Fluorescence characteristics of LucY and other MurB homologs.

<p>Ɛ, extinction coefficient</p><p>Φ_F, quantum yield</p><p>Published Φ_F for FAD and FMN are 0.032 and 0.27, respectively.</p><p>Fluorescence characteristics of LucY and other MurB homologs.</p

Alignment of active site structure of MurB enzymes.

<p>Amino acid residues are aligned within 5 Å of the isoalloxazine moiety of FAD as well as at the positions of the proposed quenching residues Trp 263 (T. caldophilus), Trp 89 (E. coli), and Tyr 42 (S. aureus) shown as spheres and sticks. All other residues and FAD are shown as sticks. Carbon is color coded as follows: LucY (green), E. coli (white), S. aureus (cyan), and T. caldophilus (yellow). Nitrogen is in blue, oxygen red, and phosphorus orange. The right figure is a 90o rotation view of the left figure around the vertical axis. Structure alignment by PyMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124272#pone.0124272.ref062" target="_blank">62</a>] is based on the isoalloxazine moiety of FAD. Inset tabulates the presence of Trp or Tyr in MurBs, with the exception of LucY. Residue numbering corresponds to species with Tyr/Trp. T. caldo for T. caldophilus.</p

Statistics for data collection and refinement.

<p>Values in parenthesis are for the highest resolution shell.</p><p>^a<i>R</i>_sym = Σ_<i>hkl</i> Σ_<i>i</i> | <i>I</i>_<i>i</i>(<i>hkl</i>)- 〈<i>I</i>(<i>hkl</i>)〉| / Σ_<i>hkl</i> Σ_<i>i</i><i>I</i>_<i>i</i>(<i>hkl</i>), where <i>I</i>_<i>i</i>(<i>hkl</i>) is the intensity of an individual measurement of the symmetry related reflection and 〈<i>I</i>(<i>hkl</i>)〉 is the mean intensity of the symmetry related reflections.</p><p>^bI/σ is defined as the ratio of averaged value of the intensity to its standard deviation.</p><p>^c<i>R</i>_cryst = Σ_<i>hkl</i> ||<i>F</i>_obs|—|<i>F</i>_calc||/ Σ_<i>hkl</i> |<i>F</i>_obs|, where <i>F</i>_obs and <i>F</i>_calc are the observed and calculated structure-factor amplitudes.</p><p>^d<i>R</i>_free was calculated as <i>R</i>_cryst using randomly selected 5% of the unique reflections that were omitted from the structure refinement.</p><p>^eRamachandran statistics indicate the percentage of residues in the most favored, additionally allowed and outlier regions of the Ramachandran diagram as defined by MOLPROBITY.</p><p>Statistics for data collection and refinement.</p