Substrate Binding Primes Human Tryptophan 2,3-Dioxygenase for Ligand Binding

The human heme enzyme tryptophan 2,3-dioxygenase (hTDO) catalyzes the insertion of dioxygen into its cognate substrate, l-tryptophan (l-Trp). Its active site structure is highly dynamic, and the mechanism of enzyme–substrate–ligand complex formation and the ensuing enzymatic reaction is not yet understood. Here we have studied complex formation in hTDO by using time-resolved optical and infrared spectroscopy with carbon monoxide (CO) as a ligand. We have observed that both substrate-free and substrate-bound hTDO coexist in two discrete conformations with greatly different ligand binding rates. In the fast rebinding hTDO conformation, there is facile ligand access to the heme iron, but it is greatly hindered in the slowly rebinding conformation. Spectroscopic evidence implicates active site solvation as playing a crucial role for the observed kinetic differences. Substrate binding shifts the conformational equilibrium markedly toward the fast species and thus primes the active site for subsequent ligand binding, ensuring that formation of the ternary complex occurs predominantly by first binding l-Trp and then the ligand. Consequently, the efficiency of catalysis is enhanced because O₂ binding prior to substrate binding, resulting in nonproductive oxidation of the heme iron, is greatly suppressed

Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

Human indoleamine 2,3-dioxygenase (hIDO) catalyzes the oxidative cleavage of the L-tryptophan (l-Trp) pyrrole ring. Catalysis is inhibited at high substrate concentrations; mechanistic details of this observation are, however, still under debate. Using time-resolved optical spectroscopy, we have analyzed the dynamics of ternary complex formation between hIDO, l-Trp, and a diatomic ligand. The physiological ligand dioxygen (O₂) was replaced by carbon monoxide to exclude enzymatic turnover. Quantitative analysis of the kinetics reveals that the ternary complex forms whenever O₂ binds first, whereas an l-Trp substrate molecule arriving prior to O₂ in the active site causes self-inhibition. Bound l-Trp prevents the ligand from approaching the heme iron and, therefore, impedes formation of the catalytically active ternary complex

Cellular Uptake of Nanoparticles by Membrane Penetration: A Study Combining Confocal Microscopy with FTIR Spectroelectrochemistry

It is well-known that nanomaterials are capable of entering living cells, often by utilizing the cells’ endocytic mechanisms. Passive penetration of the lipid bilayer may, however, occur as an alternative process. Here we have focused on the passive transport of small nanoparticles across the plasma membranes of red blood cells, which are incapable of endocytosis. By using fluorescence microscopy, we have observed that zwitterionic quantum dots penetrate through the cell membranes so that they can be found inside the cells. The penetration-induced structural changes of the lipid bilayer were explored by surface-enhanced infrared absorption spectroscopy and electrochemistry studies of model membranes prepared on solid supports with lipid compositions identical to those of red blood cell membranes. A detailed analysis of the infrared spectra revealed a markedly enhanced flexibility of the lipid bilayers in the presence of nanoparticles. The electrochemistry data showed that the overall membrane structure remained intact; however, no persistent holes were formed in the bilayers

Temperature Dependence of the Heat Diffusivity of Proteins

In a combined experimental–theoretical study, we investigated the transport of vibrational energy from the surrounding solvent into the interior of a heme protein, the sperm whale myoglobin double mutant L29W-S108L, and its dependence on temperature from 20 to 70 K. The hindered libration of a CO molecule that is not covalently bound to any part of the protein but is trapped in one of its binding pockets (the Xe4 pocket) was used as the local thermometer. Energy was deposited into the solvent by IR excitation. Experimentally, the energy transfer rate increased from (30 ps)⁻¹ at 20 K to (8 ps)⁻¹ at 70 K. This temperature trend is opposite to what is expected, assuming that the mechanism of heat transport is similar to that in glasses. In order to elucidate the mechanism and its temperature dependence, nonequilibrium molecular dynamics (MD) simulations were performed, which, however, predicted an essentially temperature-independent rate of vibrational energy flow. We tentatively conclude that the MD potentials overestimate the coupling between the protein and the CO molecule, which appears to be the rate-limiting step in the real system at low temperatures. Assuming that this coupling is anharmonic in nature, the observed temperature trend can readily be explained

Distinct amino acid motifs carrying multiple positive charges regulate membrane targeting of dysferlin and MG53

<div><p>Dysferlin (Dysf) and mitsugumin53 (MG53) are two key proteins involved in membrane repair of muscle cells which are efficiently recruited to the sarcolemma upon lesioning. Plasma membrane localization and recruitment of a Dysf fragment to membrane lesions in zebrafish myofibers relies on the presence of a short, polybasic amino acid motif, WRRFK. Here we show that the positive charges carried by this motif are responsible for this function. In mouse MG53, we have identified a similar motif with multiple basic residues, WKKMFR. A single amino acid replacement, K279A, leads to severe aggregation of MG53 in inclusion bodies in HeLa cells. This result is due to the loss of positive charge, as shown by studying the effects of other neutral amino acids at position 279. Consequently, our data suggest that positively charged amino acid stretches play an essential role in the localization and function of Dysf and MG53.</p></div

Complex RNA Folding Kinetics Revealed by Single-Molecule FRET and Hidden Markov Models

We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels–Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg²⁺ concentrations. Structures generally tend to become more compact upon addition of Mg²⁺. Some compact structures are observed to significantly depend on Mg²⁺ concentration, suggesting a tertiary fold stabilized by Mg²⁺ ions. One compact structure was observed to be Mg²⁺-independent, consistent with stabilization by tertiary Watson–Crick base pairing found in the folded Diels–Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding

Evaluation of Genetically Encoded Chemical Tags as Orthogonal Fluorophore Labeling Tools for Single-Molecule FRET Applications

Fluorescence resonance energy transfer (FRET) is a superb technique for measuring conformational changes of proteins on the single molecule level (smFRET) in real time. It requires introducing a donor and acceptor fluorophore pair at specific locations on the protein molecule of interest, which has often been a challenging task. By using two different self-labeling chemical tags, such as Halo-, TMP-, SNAP- and CLIP-tags, orthogonal labeling may be achieved rapidly and reliably. However, these comparatively large tags add extra distance and flexibility between the desired labeling location on the protein and the fluorophore position, which may affect the results. To systematically characterize chemical tags for smFRET measurement applications, we took the SNAP-tag/CLIP-tag combination as a model system and fused a flexible unstructured peptide, rigid polyproline peptides of various lengths, and the calcium sensor protein calmodulin between the tags. We could reliably identify length variations as small as four residues in the polyproline peptide. In the calmodulin system, the added length introduced by these tags was even beneficial for revealing subtle conformational changes upon variation of the buffer conditions. This approach opens up new possibilities for studying conformational dynamics, especially in large protein systems that are difficult to specifically conjugate with fluorophores

The cellular distribution of smDysf depends on the positive charge of the WRRFK motif.

<p>The cytoplasmic fraction is significantly increased upon reducing the net charge but not by polypeptide truncation on the C-terminal side of smDysf. For this plot, the cytoplasmic fluorescence of 9–15 myofibers was measured and averaged for each mutant. All data are referenced to the wildtype set to 100% and plotted as mean ± SD. The original data are provides as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202052#pone.0202052.s001" target="_blank">S1 Dataset</a>. Zebrafish embryos expressing these ten variants were injected, treated and imaged under identical conditions. The mutant sequences are grouped according to the net charge (1+– 3+) of the different motifs under physiological conditions. Significance was tested against the wildtype control smDysf (WRRFK-TM-C) by Student’s t-test (** <i>p</i> < 0.01, *** <i>p</i> < 0.001).</p

Confocal images of C2C12 myoblasts expressing turboGFP:MG53 and mutants K278A, R282A, K279A.

<p>Fluorescence was excited with a 488-nm laser. As in HeLa cells, K279A shows substantial vesicular localization in myoblasts. Scale bars, 10 μm.</p

Accumulation of smDysf at the lesion site depends on the positive charge of the WRRFK motif <i>in vivo</i>.

<p>(A) Domain structure of Dysf. The boxed fragment is smDysf, which was used to test accumulation at the lesion patch. (B) Representative images showing accumulation of smDysf and different mutants. Basic AAs are highlighted in red; arrows indicate the site of lesion. Z-line (z) and sarcolemmal (sc) regions are noted. (C-D) Corresponding kinetics of accumulation of zebrafish (C) or human (D) smDysf at the damage site, again with the basic AAs highlighted in red. The fluorescence intensity at the lesion was normalized to the one of the undamaged state. Intensity courses are averages over 9–15 damaged cells. Scale bars, 4 μm.</p