Karen Kallio

Crystal structure of the Aequorea victoria green fluorescent protein.

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded β barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein.

BACKGROUND Because of its ability to spontaneously generate its own fluorophore, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria is used extensively as a fluorescent marker in molecular and cell biology. The yellow fluorescent proteins (YFPs) have the longest wavelength emissions of all GFP variants examined to date. This shift in the spectrum is the result of a T203Y substitution (single-letter amino acid code), a mutation rationally designed on the basis of the X-ray structure of GFP S65T. RESULTS We have determined the crystal structures of YFP T203Y/S65G/V68L/S72A and YFP H148G to 2.5 and 2.6 A resolution, respectively. Both structures show clear electron density for nearly coplanar pi-pi stacking between Tyr203 and the chromophore. The chromophore has been displaced by nearly 1 A in comparison to other available structures. Although the H148G mutation results in the generation of a solvent channel to the chromophore cavity, intense fluorescence is maintained. The chromophore in the intact protein can be titrated, and the two variants have pKa values of 7.0 (YFP) and 8.0 (YFP H148G). CONCLUSIONS The observed red shift of the T203Y YFP variant is proposed to be mainly due to the additional polarizability of the pi-stacked Tyr203. The altered location of the chromophore suggests that the exact positions of nearby residues are not crucial for the chemistry of chromophore formation. The YFPs significantly extend the pH range over which GFPs may be employed as pH indicators in live cells.

Structure and mechanism of the photoactivatable green fluorescent protein.

Crystal structures of the photoactivatable green fluorescent protein T203H variant (PA-GFP) have been solved in the native and photoactivated states, which under 488 nm illumination are dark and brightly fluorescent, respectively. We demonstrate that photoactivation of PA-GFP is the result of a UV-induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form. Coupled with the T203H mutation, which stabilizes the native PA-GFP neutral chromophore, Glu222 decarboxylation yields a 100-fold contrast enhancement relative to wild-type GFP (WT). Additionally, the structures provide insights into the spectroscopic differences between WT and PA-GFP steady-state fluorescence maxima and excited-state proton transfer dynamics.

Structure of the Escherichia coli malate synthase G:pyruvate:acetyl‐coenzyme A abortive ternary complex at 1.95 Å resolution

Malate synthase, an enzyme of the glyoxylate pathway, catalyzes the condensation and subsequent hydrolysis of acetyl‐coenzyme A (acetyl‐CoA) and glyoxylate to form malate and CoA. In the present study, we present the 1.95 Å–resolution crystal structure of Escherichia coli malate synthase isoform G in complex with magnesium, pyruvate, and acetyl‐CoA, and we compare it with previously determined structures of substrate and product complexes. The results reveal how the enzyme recognizes and activates the substrate acetyl‐CoA, as well as conformational changes associated with substrate binding, which may be important for catalysis. On the basis of these results and mutagenesis of active site residues, Asp 631 and Arg 338 are proposed to act in concert to form the enolate anion of acetyl‐CoA in the rate‐limiting step. The highly conserved Cys 617, which is immediately adjacent to the presumed catalytic base Asp 631, appears to be oxidized to cysteine‐sulfenic acid. This can explain earlier observations of the susceptibility of the enzyme to inactivation and aggregation upon X‐ray irradiation and indicates that cysteine oxidation may play a role in redox regulation of malate synthase activity in vivo. There is mounting evidence that enzymes of the glyoxylate pathway are virulence factors in several pathogenic organisms, notably Mycobacterium tuberculosis and Candida albicans. The results described in this study add insight into the mechanism of catalysis and may be useful for the design of inhibitory compounds as possible antimicrobial agents.

An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V.

Wild‐type green fluorescent protein (wt‐GFP) has a prominent absorbance band centered at ∼395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited‐state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen‐bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time‐resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place ∼30 times more slowly than in wt‐GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen‐bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine‐based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt‐GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.

Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein

mPlum is a far‐red fluorescent protein with emission maximum at ∼650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far‐red emission. Ultrafast time‐resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum‐E16Q and ‐I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far‐red emission. Both the far‐red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.

The N–B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite

Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

Structure and excited-state proton transfer in the GFP S205A mutant.

To further explore excited state proton transfer (ESPT) pathways within green fluorescent protein (GFP), mutagenesis, X-ray crystallography, and time-resolved and steady-state optical spectroscopy were employed to create and study the GFP mutant S205A. In wild type GFP (wt-GFP), the proton transfer pathway includes the hydroxyl group of the chromophore, a water molecule, Ser205, and Glu222. We found that the ESPT rate constant of S205A is smaller by a factor of 20 than that of wt-GFP and larger by a factor of 2 in comparison to the ESPT rate of S205V mutant which we previously characterized. (1) High resolution crystal structures reveal that in both S205A and S205V mutants, an alternative proton transfer pathway is formed that involves the chromophore hydroxyl, a bridging water molecule, Thr203 and Glu222. The slow PT rate is explained by the long (∼3.2 Å and presumably weak) hydrogen bond between Thr203 and the water molecule, compared to the 2.7 Å normal hydrogen bond between the water molecule and Ser205 in wt-GFP. For data analysis of the experimental data from both GFP mutants, we used a two-rotamer kinetic model, assuming only one rotamer is capable of ESPT. Data analysis supports an agreement with the underlying assumption of this model.

Structures of the ligand-binding domain of Helicobacter pylori chemoreceptor TlpA: Ligand-Binding Domain of H. pylori Chemoreceptor TlpA

Bacteria use chemoreceptor proteins to sense and navigate their chemical environments. The most common class of chemoreceptors are transmembrane proteins that sense chemical cues through binding of a small‐molecule ligand to a periplasmic domain, which modulates the receptors ability to stimulate reversal of the cells flagella motors. The prevalent gastric pathogen Helicobacter pylori uses such membrane‐bound chemoreceptors, called transducer‐like proteins (Tlp), to colonize and persist within the stomach. TlpA has been implicated in sensing arginine, bicarbonate, and acid, but no experimentally determined protein structures of TlpA were available to better understand ligand binding and signal transduction. Here, we report three crystal structures of the periplasmic portion of TlpA, which contains tandem PAS/Cache domains, similar to a recently published structure of the lactate‐sensing chemoreceptor TlpC from H. pylori. These structures are the first to show a tandem PAS/Cache‐form chemoreceptor in its native homo dimer oligomer, and we identify residues that are key contributers to the dimer interface. We performed sequence analyses to identify TlpA and TlpC homologs and used residue conservation among these homologs to implicate regions important for the general tandem PAS/Cache fold, and residues specific to TlpA function. Comparisons with TlpC show that despite high similarity across the general structure, TlpA lacks the residues required to bind lactate, and instead contains a pocket almost entirely hydrophobic in nature.