Development of an Optical Zn²⁺ Probe Based on a Single Fluorescent Protein

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn²⁺. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn²⁺ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering, and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1, which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn²⁺ in HeLa cells (<i>F</i>_max/<i>F</i>_min = 2.6, <i>K</i>_d = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn²⁺ (<i>F</i>_max/<i>F</i>_min = 2.2). This sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn²⁺ function

Identifying equivalent clusters in homologous proteins allows for direct comparison of local environments.

<p>(A) A cartoon depiction of cluster of adjacent residues is shown (red circle). (B) Structural alignment of paired enzymes is shown, with PDB 1vbr in orange and 2uwf in gray. The structurally aligned residues for the paired enzymes are shown beneath. (C) Differences in atomic packing is depicted with alternate sequences shown in stick and sphere representation on PDB 2wva.</p

Evaluating the potential for epistasis.

<p>(A) The number of residues in each motif are determined for all representative thermophilic-mesophilic structure pairs and binned according to the motif size. (B) The number of residue substitutions, given as Hamming distance, in each equivalent thermophilic-mesophilic motif is determined and binned.</p

Amino acid composition for linkers shows an enrichment in serine, threonine and proline residues.

<p>(A) eukaryotic GH7/CBM1, (B) eukaryotic CBM1/GH6, (C) bacteria CBM2/GH6, and (D) bacteria GH6/CBM2. (E) A LOGO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048615#pone.0048615-Crooks1" target="_blank">[68]</a> for the linker regions from the bacterial CBM2/GH6 show low complexity resulting from the amino acid bias.</p

Linker sequences are divergent in length.

<p>The number of sequences for given lengths are shown for the (A) eukaryotic GH7/CBM1, (B) the eukaryotic CBM1/GH6, and the bacterial GH Family 6 datasets (C) CBM2/GH6 and (D) GH6/CBM2. Linker lengths are binned into five residues groups.</p

An increased occurrence of glycine residues occurs where the linkers connect to globular domains.

<p>The probability of finding a glycine residue was determined for the linker sequences in (A) the Eukaryotic GH Family 7, (B) the Eukaryotic GH Family 6, and the Bacterial GH Family 6 datasets (C) with the CBM located at the N-terminus and (D) with the CBM located at the C-terminus. Each sequence was split into 11 sections, or bins, with an approximately equal number of residues in each bin. The number of glycine residues was divided by the total number of sequence positions in each bin.</p

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

<div><p>Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. <em>O</em>-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative <em>O</em>-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, <em>O</em>-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative <em>N</em>-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of <em>N</em>-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions.</p> </div

Thermophilic enzyme clusters display closer atomic packing compared to mesophilic enzyme clusters for most enzyme pairs evaluated.

<p>(A) SASA_1.4 values for clusters from the representative thermophilic-mesophilic structure pairs are shown, with thermophilic clusters shown in red, mesophilic clusters in green and the difference, ΔSASA_1.4, in blue. Values are sorted by ΔSASA_1.4. (B) SASA_1.4 values are shown comparing clusters from the thermophilic (PDB 1a5z) and mesophilic (PDB 6ldh) lactate dehydrogenase enzymes, which have a difference in optimum activity temperature of 30°C. (C) the thermophilic (PDB 1a5z) and mesophilic (PDB 5ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 48°C, (D) and the thermophilic (PDB 1a5z) and psychrophilic (PDB 1ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 70°C.</p

The backbone can move significantly in the structurally equivalent clusters.

<p>(A) Three Cα atoms from a paired cluster are shown in red spheres (thermophilic enzyme) and purple spheres (mesophilic enzyme). The atoms are labeled a, b and c for the thermophilic enzyme and a’, b’ and c’ for the mesophilic enzyme. The Euclidian distances between Cα atoms are shown for each enzyme, with the distance differences at right. (B) The sum of the absolute values for the distance differences (red), and the average distance differences (blue) for each representative cluster are shown, sorted by summed or averaged distances.</p

Anchor residues are conserved in atomic packing.

<p>(A) The thermostable GH9 (PDB 4dod) is shown in surface representation, with anchor residues that are seen in a larger number of clusters shown in stick representation. Residues exhibiting the largest ΔSASA_1.4, which are never anchor residues, are colored red. (B) Sequence positions from 4dod are binned by the number of clusters in which they are found. The heat scale indicates ΔSASA_1.4. Importantly, blue is not observed as there are no mesophilic clusters with significantly better atomic packing relative to the matched thermophilic cluster. (C) Sequence positions from the representative set of structures are binned by the number of motifs in which they are found (x-axis), with ΔSASA_1.4 shown for each paired position (y-axis). A white symbol indicates sequence conservation, and gray indicates the sequence differs at that position.</p