Chemically stable fluorescent proteins for advanced microscopy.

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors.

We have determined the crystal structure of Clover, one of the brightest fluorescent proteins, and found that its T203H/S65G mutations relative to wild-type GFP lock the critical E222 side chain in a fixed configuration that mimics the major conformer of that in EGFP. The resulting equilibrium shift to the predominantly deprotonated chromophore increases the extinction coefficient (EC), opposes photoactivation, and is responsible for the bathochromic shift. Clover's brightness can further be attributed to a π-π stacking interaction between H203 and the chromophore. Consistent with these observations, the Clover G65S mutant reversed the equilibrium shift, dramatically decreased the EC, and made Clover photoactivatable under conditions that activated photoactivatable GFP. Using the Clover structure, we rationally engineered a non-photoactivatable redox sensor, roClover1, and determined its structure as well as that of its parental template, roClover0.1. These high-resolution structures provide deeper insights into structure-function relationships in GFPs and may aid the development of excitation-improved ratiometric biosensors.

Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites.

The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.

A phenylalanine clamp controls substrate specificity in the quorum-quenching metallo-γ-lactonase from Bacillus thuringiensis.

Autoinducer inactivator A (AiiA) is a metal-dependent N-acyl homoserine lactone hydrolase that displays broad substrate specificity but shows a preference for substrates with long N-acyl substitutions. Previously, crystal structures of AiiA in complex with the ring-opened product N-hexanoyl-l-homoserine revealed binding interactions near the metal center but did not identify a binding pocket for the N-acyl chains of longer substrates. Here we report the crystal structure of an AiiA mutant, F107W, determined in the presence and absence of N-decanoyl-l-homoserine. F107 is located in a hydrophobic cavity adjacent to the previously identified ligand binding pocket, and the F107W mutation results in the formation of an unexpected interaction with the ring-opened product. Notably, the structure reveals a previously unidentified hydrophobic binding pocket for the substrate's N-acyl chain. Two aromatic residues, F64 and F68, form a hydrophobic clamp, centered around the seventh carbon in the product-bound structure's decanoyl chain, making an interaction that would also be available for longer substrates, but not for shorter substrates. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the hydrophobic clamp, including insertion of a redox-sensitive cysteine pair, confirms the importance of this hydrophobic feature for substrate preference. Identifying the specificity determinants of AiiA will aid the development of more selective quorum-quenching enzymes as tools and as potential therapeutics.

The kinetic mechanism of S. pneumoniae DNA ligase and inhibition by adenosine-based antibacterial compounds.

The NAD-dependent DNA ligase is an excellent target for the discovery of antibacterial agents with a novel mode of action. In this work the DNA ligase from Streptococcus pneumoniae was investigated for its steady-state kinetic parameters and inhibition by compounds with an adenosine substructure. Inhibition by substrate DNA that was observed in the enzyme turnover experiments was verified by direct binding measurements using isothermal titration calorimetry (ITC). The substrate-inhibited enzyme form was identified as deadenylated DNA ligase. The binding potencies of 2-(butylsulfanyl) adenosine and 2-(cyclopentyloxy) adenosine were not significantly affected by the presence of the enzyme-bound DNA substrate. Finally, a mutant protein was prepared that was known to confer resistance to the adenosine compounds' antibacterial activity. The mutant protein was shown to have little catalytic impairment yet it was less susceptible to adenosine compound inhibition.

Antimicrobial Activity of Adenine-Based Inhibitors of NAD(+)-Dependent DNA Ligase.

The relationship between enzyme inhibition and antimicrobial potency of adenine-based NAD(+)-dependent DNA ligase (LigA) inhibitors was investigated using a strain of the Gram-negative pathogen Haemophilus influenzae lacking its major AcrAB-TolC efflux pump and the Gram-positive pathogen Streptococcus pneumoniae. To this end, biochemical inhibitors not mediating their antibacterial mode of action (MOA) via LigA were removed from the analysis. In doing so, a significant number of compounds were identified that acted via inhibition of LigA in S. pneumoniae but not in H. influenzae, despite being inhibitors of both isozymes. Deviations from the line correlating antimicrobial and biochemical potencies of LigA inhibitors with the correct MOA were observed for both species. These deviations, usually corresponding to higher MIC/IC50 ratios, were attributed to varying compound permeance into the cell.

Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity.

Optimization of adenosine analog inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3'- and 5'-substituents on the ribose. Compounds with logD values 1.5-2.5 maximized potency and maintained drug-like physical properties.

Transient non-native hydrogen bonds promote activation of a signaling protein.

Phosphorylation is a common mechanism for activating proteins within signaling pathways. Yet, the molecular transitions between the inactive and active conformational states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of a signaling protein, nitrogen regulatory protein C (NtrC), by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding and show that only a rarely populated, pre-existing active conformation is energetically stabilized by phosphorylation. Using nuclear magnetic resonance (NMR) dynamics, we test an atomic scale pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009). The data show that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results unravel atomistic details of native-state protein energy landscapes by expanding the knowledge about ground states to transition landscapes.