32 research outputs found
Reaction-Based Probes for Imaging Mobile Zinc in Live Cells and Tissues
Chelatable, or mobile, forms of zinc play critical signaling roles in numerous biological processes. Elucidating the action of mobile Zn(II) in complex biological environments requires sensitive tools for visualizing, tracking, and manipulating Zn(II) ions. A large toolbox of synthetic photoinduced electron transfer (PET)-based fluorescent Zn(II) sensors are available, but the applicability of many of these probes is limited by poor zinc sensitivity and low dynamic ranges owing to proton interference. We present here a general approach for acetylating PET-based probes containing a variety of fluorophores and zinc-binding units. The new sensors provide substantially improved zinc sensitivity and allow for incubation of live cells and tissue slices with nM probe concentrations, a significant improvement compared to the μM concentrations that are typically required for a measurable fluorescence signal. Acetylation effectively reduces or completely quenches background fluorescence in the metal-free sensor. Binding of Zn(II) selectively and quickly mediates hydrolytic cleavage of the acetyl groups, providing a large fluorescence response. An acetylated blue coumarin-based sensor was used to carry out detailed analyses of metal binding and metal-promoted acetyl hydrolysis. Acetylated benzoresorufin-based red-emitting probes with different zinc-binding sites are effective for sensing Zn(II) ions in live cells when applied at low concentrations (∼50–100 nM). We used green diacetylated Zinpyr1 (DA-ZP1) to image endogenous mobile Zn(II) in the molecular layer of mouse dorsal cochlear nucleus (DCN), confirming that acetylation is a suitable approach for preparing sensors that are highly specific and sensitive to mobile zinc in biological systems.National Institutes of Health (U.S.) (NIH grant GM065519)National Institutes of Health (U.S.) (NIH grant R01-DC007905)National Institutes of Health (U.S.) (NIH Fellowship (F32- EB019243))National Institutes of Health (U.S.) (NIH Fellowship (T32-DC011499))National Institutes of Health (U.S.) (NIH Fellowship (F32-DC013734)
Influence of Active Site Location on Catalytic Activity in <i>de Novo</i>-Designed Zinc Metalloenzymes
While metalloprotein design has now
yielded a number of successful
metal-bound and even catalytically active constructs, the question
of where to put a metal site along a linear, repetitive sequence has
not been thoroughly addressed. Often several possibilities in a given
sequence may exist that would appear equivalent but may in fact differ
for metal affinity, substrate access, or protein dynamics. We present
a systematic variation of active site location for a hydrolytically
active ZnHis<sub>3</sub>O site contained within a <i>de novo</i>-designed three-stranded coiled coil. We find that the maximal rate,
substrate access, and metal-binding affinity are dependent on the
selected position, while catalytic efficiency for <i>p</i>-nitrophenyl acetate hydrolysis can be retained regardless of the
location of the active site. This achievement demonstrates how efficient,
tailor-made enzymes which control rate, p<i>K</i><sub>a</sub>, substrate and solvent access (and selectivity), and metal-binding
affinity may be realized. These findings may be applied to the more
advanced <i>de novo</i> design of constructs containing
secondary interactions, such as hydrogen-bonding channels. We are
now confident that changes to location for accommodating such channels
can be achieved without location-dependent loss of catalytic efficiency.
These findings bring us closer to our ultimate goal of incorporating
the secondary interactions we believe will be necessary in order to
improve both active site properties and the catalytic efficiency to
be competitive with the native enzyme, carbonic anhydrase
Hydrolytic catalysis and structural stabilization in a designed metalloprotein
Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions – a Zn(II) ion which is important for catalytic activity and a Hg(II) ion which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate hydrolysis (pNPA) to within ~100-fold of the efficiency of human carbonic anhydrase (CA)II and is at least 550-fold better than comparable synthetic complexes. Similarly, CO(2) hydration occurs with an efficiency within ~500-fold of CAII. While histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO(2) hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme uncovers necessary design features for future metalloenzymes containing one or more metals
Biomimetic chemistry for the next fifty years: De novo design of zinc and copper proteins
A Crystallographic Examination of Predisposition versus Preorganization in de Novo Designed Metalloproteins
Preorganization and predisposition
are important molecular recognition
concepts exploited by nature to obtain site-specific and selective
metal binding to proteins. While native structures containing an MS<sub>3</sub> core are often unavailable in both apo- and holo-forms, one
can use designed three-stranded coiled coils (3SCCs) containing tris-thiolate
sites to evaluate these concepts. We show that the preferred metal
geometry dictates the degree to which the cysteine rotamers change
upon metal complexation. The Cys ligands in the apo-form are preorganized
for binding trigonal pyramidal species (Pb(II)S<sub>3</sub> and As(III)S<sub>3</sub>) in an <i>endo</i> conformation oriented toward
the 3SCC C-termini, whereas the cysteines are predisposed for trigonal
planar Hg(II)S<sub>3</sub> and 4-coordinate Zn(II)S<sub>3</sub>O structures,
requiring significant thiol rotation for metal binding. This study
allows assessment of the importance of protein fold and side-chain
reorientation for achieving metal selectivity in human retrotransposons
and metalloregulatory proteins
A Crystallographic Examination of Predisposition versus Preorganization in de Novo Designed Metalloproteins
Protein Design: Toward Functional Metalloenzymes
The scope of this Review is to discuss the construction of metal sites in designed protein scaffolds. We categorize the effort of designing proteins into redesign, which is to rationally engineer desired functionality into an existing protein scaffold,(1-9) and de novo design, which is to build a peptidic or protein system that is not directly related to any sequence found in nature yet folds into a predicted structure and/or carries out desired reactions.(10-12) We will analyze and interpret the significance of designed protein systems from a coordination chemistry and biochemistry perspective, with an emphasis on those containing constructed metal sites as mimics for metalloenzymes
A far-red emitting probe for unambiguous detection of mobile zinc in acidic vesicles and deep tissue
Imaging mobile zinc in acidic environments remains challenging because most small-mol. optical probes display pH-dependent fluorescence. Here we report a reaction-based sensor that detects mobile zinc unambiguously at low pH. The sensor responds reversibly and with a large dynamic range to exogenously applied Zn2+ in lysosomes of HeLa cells, endogenous Zn2+ in insulin granules of MIN6 cells, and zinc-rich mossy fiber boutons in hippocampal tissue from mice. This long-wavelength probe is compatible with the green-fluorescent protein, enabling multicolor imaging, and facilitates visualization of mossy fiber boutons at depths of \textgreater100 μm, as demonstrated by studies in live tissue employing two-photon microscopy
A far-red emitting probe for unambiguous detection of mobile zinc in acidic vesicles and deep tissue
Imaging mobile zinc in acidic environments remains challenging because most small-molecule optical probes display pH-dependent fluorescence. Here we report a reaction-based sensor that detects mobile zinc unambiguously at low pH. The sensor responds reversibly and with a large dynamic range to exogenously applied Zn²⁺ in lysosomes of HeLa cells, endogenous Zn²⁺ in insulin granules of MIN6 cells, and zinc-rich mossy fiber boutons in hippocampal tissue from mice. This long-wavelength probe is compatible with the green-fluorescent protein, enabling multicolor imaging, and facilitates visualization of mossy fiber boutons at depths of >100 μm, as demonstrated by studies in live tissue employing two-photon microscopy.National Institute of General Medical Sciences (U.S.) (GM065519