3 research outputs found
Ultrafast Proton Shuttling in <i>Psammocora</i> Cyan Fluorescent Protein
Cyan, green, yellow, and red fluorescent
proteins (FPs) homologous
to green fluorescent protein (GFP) are used extensively as model systems
to study fundamental processes in photobiology, such as the capture
of light energy by protein-embedded chromophores, color tuning by
the protein matrix, energy conversion by Förster resonance
energy transfer (FRET), and excited-state proton transfer (ESPT) reactions.
Recently, a novel cyan fluorescent protein (CFP) termed psamFP488
was isolated from the genus <i>Psammocora</i> of reef building
corals. Within the cyan color class, psamFP488 is unusual because
it exhibits a significantly extended Stokes shift. Here, we applied
ultrafast transient absorption and pump–dump–probe spectroscopy
to investigate the mechanistic basis of psamFP488 fluorescence, complemented
with fluorescence quantum yield and dynamic light scattering measurements.
Transient absorption spectroscopy indicated that, upon excitation
at 410 nm, the stimulated cyan emission rises in 170 fs. With pump–dump–probe
spectroscopy, we observe a very short-lived (110 fs) ground-state
intermediate that we assign to the deprotonated, anionic chromophore.
In addition, a minor fraction (14%) decays with 3.5 ps to the ground
state. Structural analysis of homologous proteins indicates that Glu-167
is likely positioned in sufficiently close vicinity to the chromophore
to act as a proton acceptor. Our findings support a model where unusually
fast ESPT from the neutral chromophore to Glu-167 with a time constant
of 170 fs and resulting emission from the anionic chromophore forms
the basis of the large psamFP488 Stokes shift. When dumped to the
ground state, the proton on neutral Glu is very rapidly shuttled back
to the anionic chromophore in 110 fs. Proton shuttling in excited
and ground states is a factor of 20–4000 faster than in GFP,
which probably results from a favorable hydrogen-bonding geometry
between the chromophore phenolic oxygen and the glutamate acceptor,
possibly involving a short hydrogen bond. At any time in the reaction,
the proton is localized on either the chromophore or Glu-167, which
implies that most likely no low-barrier hydrogen bond exists between
these molecular groups. This work supports the notion that proton
transfer in biological systems, be it in an electronic excited or
ground state, can be an intrinsically fast process that occurs on
a 100 fs time scale. PsamFP488 represents an attractive model system
that poses an ultrafast proton transfer regime in discrete steps.
It constitutes a valuable model system in addition to wild type GFP,
where proton transfer is relatively slow, and the S65T/H148D GFP mutant,
where the effects of low-barrier hydrogen bonds dominate
Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach
Higher-plant
Rubisco activase (Rca) plays a critical role in regulating
the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).
In vitro, Rca is known to undergo dynamic assembly–disassembly
processes, with several oligomer stoichiometries coexisting over a
broad concentration range. Although the hexamer appears to be the
active form, changes in quaternary structure could play a role in
Rubisco regulation. Therefore, fluorescent labels were attached to
the C-termini of spinach β-Rca, and the rate of subunit mixing
was monitored by measuring energy transfer as a function of nucleotide
and divalent cation. Only dimeric units appeared to exchange. Poorly
hydrolyzable substrate analogues provided locked complexes with high
thermal stabilities (apparent <i>T</i><sub>m</sub> = 60
°C) and an estimated <i>t</i><sub>1/2</sub> of at least
7 h, whereas ATP-Mg provided tight assemblies with <i>t</i><sub>1/2</sub> values of 30–40 min and ADP-Mg loose assemblies
with <i>t</i><sub>1/2</sub> values of <15 min. Accumulation
of ADP to 20% of the total level of adenine nucleotide substantially
accelerated equilibration. An initial lag period was observed with
ATP·Mg, indicating inhibition of subunit exchange at low ADP
concentrations. The ADP <i>K</i><sub>i</sub> value was estimated
to exceed the <i>K</i><sub>m</sub> for ATP (0.772 ±
96 mM), suggesting that the equilibration rate is a function of the
relative contributions of high- and low-affinity states. C-Terminal
cross-linking generated covalent dimers, required the N-terminal extension
to the AAA+ domain, and provided evidence of different classes of
sites. We propose that oligomer reorganization may be stalled during
periods of high Rubisco reactivation activity, whereas changes in
quaternary structure are stimulated by the accumulation of ADP at
low light levels
ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations
We
report a fluorescence correlation spectroscopy (FCS) study of
the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase
responsible for activation of inhibited Rubisco complexes for biological
carbon fixation. A thermodynamic characterization of simultaneously
populated oligomeric states appears critical in understanding Rca
structure and function. Using cotton β-Rca, we demonstrate that
apparent diffusion coefficients vary as a function of concentration,
nucleotide, and cation. Using manual fitting procedures, we provide
estimates for the equilibrium constants for the stepwise assembly
and find that in the presence of ATPγS, the <i>K</i><sub>d</sub> for hexamerization is 10-fold lower than with ADP (∼0.1
vs ∼1 μM). Hexamer fractions peak at 30 μM and
dominate at 8–70 μM Rca, where they comprise 60–80%
of subunits with ATPγS, compared with just 30–40% with
ADP. Dimer fractions peak at 1–4 μM Rca, where they comprise
15–18% with ATPγS and 26–28% with ADP. At 30 μM
Rca, large aggregates begin to form that comprise ∼10% of total
protein with ATPγS and ∼25% with ADP. FCS data collected
on the catalytically impaired WalkerB-D173N variant in the presence
of ATP provided strong support for these results. Titration with free
magnesium ions lead to the disaggregation of larger complexes in favor
of hexameric forms, suggesting that a second magnesium binding site
with a <i>K</i><sub>d</sub> value of 1–3 mM mediates
critical subunit contacts. We propose that closed-ring toroidal hexameric
forms are stabilized by binding of Mg·ATP plus Mg<sup>2+</sup>, whereas Mg·ADP promotes continuous assembly to supramolecular
aggregates such as spirals