Directory UMM :Data Elmu:jurnal:P:PlantScience:Plant Science_BioMedNet:141-160:
trends in plant science
update
28 Rathcke, B. (1988) Interactions for pollination
among coflowering shrubs, Ecology 69, 446–457
29 Schürch, S. et al. Effects of ants on the
reproductive success of Euphorbia cyparissias
and associated pathogenic rust fungi, Oikos
(in press)
30 Primack, R.B. (1985) Longevity of individual
flowers, Annu. Rev. Ecol. Syst. 16, 15–38
31 Ashman, T-L. and Schoen, D.J. (1994) How
long should flowers live? Nature 371, 788–791
32 Williamson, G.B. (1982) Plant mimicry:
evolutionary constraints, Biol. J. Linn. Soc. 18, 49–58
33 Waser, N.M. (1983) Competition for pollination
and floral character differences among sympatric
plant species: a review of evidence, in Handbook
of Experimental Pollination Biology (Jones, C.E.
and Little, R.J., eds), pp. 277–292, Van Nostrand
Reinhold
34 Murcia, C. and Feinsinger, P. (1996) Interspecific
pollen loss by hummingbirds visiting flower
mixtures: effects of floral architecture, Ecology
77, 550–560
35 Roy, B.A. and Raguso, R.A. (1997) Olfactory
versus visual cues in a floral mimicry system,
Oecologia 109, 414–426
36 Raguso, R.A. and Roy, B.A. (1998) ‘Floral’ scent
production by Puccinia rust fungi that mimic
flowers, Mol. Ecol. 7, 1127–1136
37 Waser, N.M. (1978) Competition for
hummingbird pollination and sequential
flowering in two Colorado wildflowers, Ecology
59, 934–944
38 Armbruster, W.S. and McGuire, A.D. (1991)
Experimental assessment of reproductive
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interactions between sympatric Aster and
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Thomson, J.D. (1980) Skewed flowering
distributions and pollinator attraction, Ecology
61, 572–579
Dafni, A. et al. (1990) Red bowl-shaped flowers:
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Smithson, A. and Macnair, M.R. (1997) Negative
frequency-dependent selection by pollinators on
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deceit and floral mimesis in Thelymitra antennifera
(Orchidaceae), Plant Syst. Evol. 158, 11–22
Heinrich, B. (1975) Energetics of pollination,
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Charlesworth, B. (1994) The genetics of adaptation:
lessons from mimicry, Am. Nat. 144, 839–847
Feinsinger, P. et al. (1986) Floral neighborhood
and pollination success in four hummingbirdpollinated cloud forest plant species, Ecology 67,
449–464
Dukas, R. (1987) Foraging behavior of three bee
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Caged peptides and proteins:
new probes to study polypeptide
function in complex biological systems
Now that the genomes of Saccharomyces
cerevisiae and Caenorhabditis elegans have
been sequenced, and the sequencing of the
Arabidopsis genome is well under way, cell
biologists are faced with the daunting challenge of establishing the function and mode of
action of thousands of gene products. The ultimate goal of these studies will be to understand how the collective functions of these
proteins and other biomolecules are responsible
for life itself. Among the new tools that need to
be developed to meet these challenges are optical probes and imaging technologies: these
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August 1999, Vol. 4, No. 8
must be capable of identifying and mapping
the interactions and activities of specific proteins
and measuring their associated kinetic parameters with high spatial and temporal resolution.
Light-directed activation of caged (inactive)
compounds should be a valuable technique for
such investigations because it can be used to
manipulate the activity of specific biomolecules in cells (for a comprehensive review of
the methodology involved see Ref. 1). This
technique involves a biologically inert, caged
compound that is specifically and rapidly (ns
to ms) perturbed within a cell using a pulse of
49 Kevan, P.G. (1983) Floral colors through the
insect eye: what they are and what they mean, in
Handbook of Experimental Pollination Biology
(Jones, C.E. and Little, R.J., eds), pp. 3–30,
Van Nostrand Reinhold
50 Chittka, L. et al. (1994) Ultraviolet as a
component of flower reflections, and the colour
perception of hymenoptera, Vision Res. 34,
1489–1508
51 Dafni, A. and Kevan, P.G. (1997) Flower size
and shape: implications in pollination,
Isr. J. Plant Sci. 45, 201–211
52 Dafni, A. (1983) Pollination of Orchis caspia –
a nectarless plant which deceives the pollinators
of nectariferous species from other plant families,
J. Ecol. 71, 467–474
53 Kullenberg, B. (1961) Studies in Ophrys
pollination, Zool. Bidr. Uppsala 34, 1–349
54 Roy, B.A. (1996) A plant pathogen influences
pollinator behavior and may influence
reproduction of non hosts, Ecology 77, 2445–2457
Bitty A. Roy* and Alex Widmer are at the
Geobotanical Institute, Swiss Federal
Institute of Technology (ETH),
Zürichberstr. 38, CH-8044 Zürich,
Switzerland.
*Author for correspondence
(tel 141 01 632 7787;
fax 141 01 632 1215;
e-mail [email protected]).
near ultraviolet light. The effects of a controlled
and local (sub-micron) concentration or activity
jump can then be recorded using timeresolved imaging of a probe of the perturbed
reaction. Specific modulation of protein activity
with light can initiate a specific cell process
that might be used to elucidate the function of
the protein, the kinetics of its activity and the
mechanism of its action (for example, activation
of myosin at the cell cortex and motility).
Over the past 20 years several caged ligands
and substrates have been used to investigate
the molecular basis of intracellular processes,
including muscle contraction2, synaptic transmission3, intracellular signaling4 and motility5.
Unfortunately, small ligands often activate
multiple cellular processes, or can be metabolized to other active species. Caged conjugates
of peptides and proteins are often better suited
for manipulating the activity of a specific protein within a cell6,7. The principle of lightdirected activation of caged polypeptides, and
the potential applications of this technique for
studying the function of specific proteins in
cellular processes are illustrated in Fig. 1.
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(99)01452-1
trends in plant science
update
Functional and spectroscopic
characterization of caged polypeptides
Caged polypeptides should be subjected to a
rigorous biochemical and spectroscopic characterization before and after photoactivation.
These studies should establish the stability of
the caged amino acid residue, the efficiency of
the photoactivation reaction and demonstrate
that photoactivation proceeds without deleterious secondary reactions. It is also important to confirm that the photoproducts of the
photoactivation reaction are benign – this condition has been demonstrated in several applications of light-directed activation of caged
polypeptides in live cells6,8–10.
(a)
Caged peptide Protein
Active peptide
Peptide binding
hu
Caged protein
Ligand
Active protein
Ligand binding
hu
(b)
Cell adhesion protein
Caged peptides
Photolabile reagents were widely used by peptide chemists during the 1970s to protect amino,
phenolic tyrosine and carboxyl groups of amino
acids11. More recently, these reagents have
been used to direct the syntheses of surfacebased, spatially addressable peptide libraries
that can be used to identify novel proteins and
drugs by high throughput screening of chemical and biological libraries12. Peptides specifically caged on arginine10 or tyrosine6,13,
cysteine or thiophosphate groups14 (Fig. 2)
have been described and should prove useful
in studying static or kinetic aspects of specific
protein activity in vivo. A major challenge is
to design a caged peptide that specifically and
potently interferes with the protein or process
under study only after photoactivation.
Caged peptide inhibitors of calmodulin
and myosin light chain kinase
In a comprehensive study involving the development of novel, caged peptides to probe the
function of calmodulin6, the timing and localization of calcium-bound calmodulin (CAM)
activity was assessed in the context of the
motile behavior of eosinophil cells. Several
proteins involved in calcium ion signaling
pathways are activated as a consequence of
CAM binding to specific sequences, including
myosin light chain kinase (MLCK), which
activates the contraction of actomyosin complexes via phosphorylation of myosin light
chain (MLC) (Fig. 3). A 20 amino acid peptide representing the target sequence in
MLCK binds tightly to CAM and inhibits its
signaling function in cell motility. A caged
peptide is designed to act as an inhibitor of
CAM only on removal by light of a protection
group from a single tyrosine residue (caged
RS-20; Fig. 3). The photolabile group is optimized for its rapid photo-release, high quantum yield and the low toxicity of its
photoproduct (Fig. 2). Caged RS-20 binds
CAM 50 times less tightly than the unprotected peptide and is effectively inactive when
assayed in smooth muscle cells known to be
dependent on CAM function. In subsequent
Cell wall
Plasma membrane
Actin and actin-binding
proteins
Signal
transduction
membrane
receptor
Gene
regulation
transcription
factors
Cytoplasm
Cell physiology
Metabolic enzymes
Nucleus
Trends in Plant Science
Fig. 1. Principle of light-directed activation of caged polypeptides. (a) A peptide that inhibits
or activates the activity of a protein is modified with a photolabile group at a specific residue
to block its ability to bind to its target protein. Near ultraviolet irradiation removes the protection
group and generates an active peptide that binds to the target protein. Similarly, a caged protein
can be created by labeling an amino acid in the protein that is essential for its activity (e.g. at
a substrate or ligand-binding site). (b) Photoactivation of microinjected caged polypeptides at
specific cellular locations can be used to investigate the function and kinetics of specific proteins
in processes that might include: membrane receptor signaling, calcium signaling, regulation
of cell adhesion at focal contacts, actin polymerization, cellular energetics or metabolism, and
studies, caged RS-20 has been microinjected
into eosinophil cells and the majority of microinjected cells exhibit a normal motile behavior before exposure to light, but, within
seconds of being irradiated, they round-up and
stop moving because of the effects of the
increased intracellular level of active RS-20.
Irradiated cells resume their motile behavior
at least 50 s after triggering the RS-20
concentration jump, indicating that the
RS-20-mediated inhibition of motility is
reversible. Control experiments show no change
in motile behavior. To demonstrate that the
cessation of motility had been caused by the
inhibition of CAM signaling to MLCK, and
not through other proteins, a caged peptide
inhibitor of MLCK was designed, based on a
peptide sequence in the auto-inhibitory
domain of MLCK (caged LSM-1; Fig. 3).
Functional in vitro assays indicate that caged
LSM-1 inhibits MLCK activity only after
photo-activation. Cells loaded with caged
LSM-1 are fully motile, but again, after photoactivation they round up and stop moving.
Because inhibition of myosin II activity
depends on blocking CAM-MLCK activity
and also on the removal of phosphates by
phosphatase, the timing of the inhibition indicates that phosphate turnover on the light
chain of myosin II must occur with seconds.
These data therefore support the conclusion
that CAM-MLCK-mediated phosphorylation
of myosin II and de-phosphorylation by protein phosphatases are sufficiently rapid to
allow eosinophil cells to respond to the rapid
changes in extracellular gradients of chemoattractant, or fluctuations in intracellular calcium ions (Fig. 3). Because several of the
August 1999, Vol. 4, No. 8
331
trends in plant science
update
(a)
H
CO
H
OCOCl
H
Lysylpolypeptide
NO2
H3CO
H
hu
NH
NO2
NO
+ NH2(CH2)4
H3CO
Tyrosinylpeptide
Br
NO2
OCH3
OH
CO
H
HOOC
CH2 CH
O
HOOC
Fmoctyrosine
CH
+ CO2
OCH3
H
H3COOC
CO
NH
H3CO
OCH3
(b)
O
OCONH (CH2)4 CH
H
O
hu
NH
NO2
NO +
CH2
NH CH CO
(c)
H
CO
H
Br
H
H
Cysteinylpolypeptide
NO2
H3CO
H
CH2 CH
S
NO2
NO
O
CH2
OCH3
Cl
O CO N
+
N(CH3)2
NO2
(f)
NH-(CH2)5CONH-CH2
O
H
N O C
O
H3CO
NH
OCH3
(e)
-
+ HS CH2 CH
H3CO
OCH3
H
CO
hu
NH
H3CO
(d)
O
H3C
H
Biotin
Br
O
NO2
OCH3
O
H
O
NO2
C O N
O
O
Trends in Plant Science
Fig. 2. Chemical strategies used to prepare caged peptides and caged proteins. (a) The e-amino group of lysine can be derivatized with
6-nitroveratryloxycarbonyl chloride to form the corresponding carbamate between a pH value of 8.0 and 9.5 at room temperature. Irradiation of
the carbamate with 365 nm light results in the de-protection of the e-amino group with the concomitant release of carbon dioxide and 3,4dimethoxy-2-nitrosobenzaldehyde. (b) The phenolic oxygen of tyrosine can be protected by alkylation via 2-bromo-a-carboxyl-2-nitrophenyl
methyl ester. Irradiation of the caged peptide rapidly releases the unprotected tyrosine peptide and a relatively non-reactive photoproduct. (c) The
thiol group of a cysteine residue can be protected with 4,5-dimethoxy-2-nitrobenzyl bromide or with 2-bromo-2-(2-nitrophenyl) acetic acid. The
products of photolysis are the free thiol group of cysteine and a potentially thiol-reactive photoproduct. A series of bifunctional photocleavable
reagents have also been used to prepare caged proteins (d and e), and a caged biotin (f).
signaling components in the transduction pathway that leads to eosinophil motility are known
(Fig. 3), and many of these biomolecules can
be caged, it should be possible to use the photoactivation technique to completely describe
the intracellular kinetics of this pathway.
Caged proteins
A caged protein is defined as a protein whose
activity is inhibited following covalent modification of an essential amino acid with a photolabile group – irradiation of a caged protein
with near ultraviolet light removes the protection group from the conjugate and restores its
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August 1999, Vol. 4, No. 8
activity7,15. For example, a caged G-actin conjugate has been described in which lysine
residues essential for actin polymerization
were modified with 6-nitroveratryloxycarbonyl chloride7. Ultraviolet irradiation of
caged actin triggers a photoactivation reaction that liberates native, polymerizationcompetent G-actin, carbon dioxide and
3,4-dimethoxy-2-nitrosobenzaldehyde (Fig. 2).
The same approach has been used to prepare
caged conjugates of profilin16 and the transcription factor GAL418 (Ref. 9). The same
photolabile group attached to lysine residues
of a monoclonal antibody has been used to
prepare a caged antibody that could only bind
its antigen after photoactivation17. A caged
heavy meromyosin (HMM) has also been prepared by labeling an essential cysteine residue
with the photolabile reagent, 4,5-dimethoxy2-nitrobenzenzylbromide15 (Fig. 2), whereas a
related molecule has been used to prepare a
caged pore-forming protein18. In general, cysteine residues are more selectively labeled
compared with lysine because of their lower
frequency in proteins and the higher reactivity of the thiol group at pH 6–8. In addition,
cysteine-mutagenesis can be employed to
design a protein for better caging chemistry
trends in plant science
update
and photoactivation18. Another method to prepare caged proteins is to use photocleavable
crosslinking reagents (Fig. 2). Here, the protein is first modified at a lysine or cysteine
residue using the photocleavable reactive
group of a bifunctional reagent. If the protein
activity is caged after this reaction, then the
second reactive group of the crosslinking
reagent is used to target the caged protein to
another biomolecule, or to a specific location,
via an antibody for later photo-activation19–21.
Photocleavable crosslinking reagents have
been used in this way to prepare caged ricin
for targeting to specific cancer cells19: a fluorescent caged actin monomer21 (Fig. 1) and a
caged actin dimer20 (Fig. 2). More specialized
methods to prepare caged proteins include the
use of photocleavable cinnamate ester substrates to target a reactive serine residue in
thrombin22, or by employing in vitro translation techniques (as discussed here).
Caged myosin
Motor proteins, powered by the hydrolysis of
ATP, are present in all cell types, but the
mechanisms that control the organization and
function of active motor proteins are poorly
understood. Caged motor proteins would be
extremely useful for unravelling spatial and
temporal aspects of motor protein function23.
This approach requires the development of
caged motor proteins that can be incorporated
into such systems, or the development of
methods for modifying endogenous motors with
a caging reagent. Progress towards these goals
has been achieved in studies on light-directed
activation of a caged myosin sub-fragment,
HMM (Refs 15,24). The function of myosin II,
a chemo-mechanical enzyme, is mediated by
coupling two different activities in the motor
molecule. The first, an F-actin-activated Mg2+ATPase, is located in the head region, whereas
the second uses free energy derived from ATP
hydrolysis to drive a stroke of the long a-helix
in the neck that produces a 5 nm translation of
the bound actin filament.
Recent investigations aimed at understanding
the molecular basis of myosin function have
focused on the region around Cys707, which is
close to the fulcrum of the long a-helix. Most
chemical modifications of myosin at this residue
elevate the Mg2+-activated ATPase activity of
the conjugate and inhibit its ability to move actin
filaments (as measured in an in vitro motility
assay sensitive to the chemo-mechanical
coupling25). To elaborate on the importance of the
hinge region in coupling the two activities of
HMM: 4,5-dimethoxy-2-nitrobenzenzylbromide
has been employed to specifically modify
Cys707. As expected, the solution-based Mg2+activated ATPase activity of this conjugate is
higher than native HMM. The functional activity of HMM is determined using a motility assay
on the following surface-bound preparations:
Rho
(a)
MLC
IP3
2+
Ca
CAM
MLCK
MLC
Rho kinase
MBS P
MBS
MLCP
MLCP
MBS
P
Phosphatase?
Actomyosin ATPase
Small GTPase?
(b)
Caged RS-20
+
NH3 -ARRKYQKTGHAVRAIGRLSS-CO2
-
Caged tyrosine
Caged LSM
+
NH3 -LSKDRMKKYMARR-CO2
-
(c)
Net force
Net force
Myosin II
Phosphorylated
myosin II filaments
Actin filaments
Ca2+ CAM
ATP
Pi
Net force
MLCK
ADP
MLCP
Trends in Plant Science
Fig. 3. Essential biomolecules and their sequence in the calcium-mediated signaling pathway
regulating actomyosin contraction during cell motility. (a) Caged biomolecules that could be
used to further define this signaling pathway include calcium ions, inositol triphosphate and
polypeptide inhibitors or activators of Rho kinase, the myosin-binding subunit (MBS) of
myosin light chain phosphatase (MLCP), MLCP and myosin II. (b) Caged RS-20 was prepared by introducing a photolabile tyrosine at position 5 in the myosin light chain kinase
(MLCK) sequence targeted by calmodulin. Photoactivation of the caged peptide generates the
deprotected RS-20, which inhibits calmodulin 50 times more effectively than caged RS-20.
Similarly, caged LSM-1 was prepared by introducing a photolabile tyrosine residue at position nine in a MLCK auto-inhibitory sequence. (c) Schematic of a eukaryotic cell undergoing locomotion. One process thought to be important is the phosphorylation of the myosin
light chain (MLC) of myosin II by Ca2+-calmodulin (CAM)-dependent MLCK. The timing of
this process was examined by rapid, localized photorelease of caged peptides in actively
motile cells6. Cessation of motility owing to inhibition of CAM or MLCK occurred within
10 s, demonstrating that turnover of active phosphorylated myosin II occurs in this time
domain. The organization of active CAM and MLCK can be established with these caged
peptides if peptide diffusion is restricted.
• Native HMM.
• HMM modified on Cys707 with the caged
reagent (caged HMM).
• Caged HMM after irradiation with a pulse
of near ultraviolet light.
Native HMM supports the movement of actin
filaments at a velocity of 3–4 mm/s, whereas
no movement is observed with caged HMM.
HMM is therefore caged in the sense that the
two activities in the conjugate are uncoupled.
However, irradiation with a short pulse of
365 nm light removes the protection group
from Cys707, which results in most actin filaments sliding in the image field at an average
velocity of 2 mm/s. These, and other studies,
suggest that modifying Cys707 with chemical
groups physically hinders the progression of
an ATP hydrolysis-dependent conformational
transition from the head to the neck region that
ordinarily leads to the power stroke of the
lever arm. Light-directed removal of the protection group from Cys707 relieves this physical constraint and restores chemo-mechanical
coupling within HMM with a time constant
of 22 ms (Ref. 15). Preliminary studies have
shown that caged myosin can also be prepared
in functional muscle fibers treated with 4,5dimethoxy-2-nitrobenzenzylbromide. Force
generation is severely impaired in the caged
myosin-containing muscle fibers – yet, as in
August 1999, Vol. 4, No. 8
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trends in plant science
update
the previous study, chemomechanical force
production recovers in the fiber about 20 ms after
removing protection groups with near ultraviolet light24. Further in vivo photoactivation experiments are planned to obtain a better understanding of how the structural dynamics in the
myosin II molecule are related to muscle contraction. This question could be addressed by
recording time-resolved measurements of force
in conjunction with time-resolved X-ray structural analysis before and after photoactivation
of the caged myosin in the muscle fiber. Lightdirected activation of caged non-muscle myosin
isoforms microinjected into motile cells is also
feasible and these studies should prove useful in
defining the kinetics and functional properties
of this protein during motility and cytokinesis.
In vitro translation systems
Recently, methods have been described to prepare caged proteins by supplementing in vitro
translation systems with cRNA containing a
rare or nonsense codon and the complimentary anti-codon tRNA charged with a caged
amino acid. Judicious choice of the site of
incorporation of a caged amino acid can lead
to the expression of a caged protein whose
activity can be triggered with a pulse of near
ultraviolet light26. This strategy has been used
to replace the active site aspartate residue of
lysozyme with caged aspartate26. Caged proteins made by in vitro translation systems are
usually only characterized by activity measurements because of the limited amount of
caged protein synthesized27. A similar
approach has been used to incorporate sitespecific photolabile amino acids into ion channels expressed in Xenopus oocytes28, including
a caged nicotinic acetylcholine receptor27.
Here, oocytes were co-injected with a caged
tyrosine-charged tRNA containing the nonsense anticodon (CUA) and a cRNA of the
a-subunit of nicotinic acetylcholine receptor,
in which a single nonsense codon (UAG)
replaces the tyrosine codon. Full inactivation
of receptor channel activity requires masking
two specific tyrosine residues (one per
a-subunit) at a surface loop of the channel
opening. The channel activity of the membrane-incorporated (caged) receptor complex
is recorded with ms time resolution before
and after irradiation of the egg with ms pulses
of near ultraviolet light. This study demonstrated that a substantial fraction of acetylcholine receptor activity can be rapidly
generated after irradiating the oocyte with
several short pulses of near ultraviolet light27.
This powerful approach extends the applications of light-directed activation of protein
activity to proteins that might be difficult to
purify or to specifically label with a caged
reagent, or for caged proteins that might be
difficult to reconstitute into model or natural
membrane systems.
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August 1999, Vol. 4, No. 8
Summary
Light-directed activation of caged compounds
has emerged as a powerful and general technique
to address the function and mechanisms of protein activity in complex molecular environments. Photoactivation of biomolecules from
ions to second messengers and from metabolites
to polypeptides can be used to reveal information
about the timing and spatial regulation of the
distribution and activity of the biomolecule in
cells and tissues. More recently, reagents and
labeling techniques have been introduced to cage
the activity of almost any biologically active
polypeptide by in vitro or in vivo labeling of
essential amino acids. These include lysine, tyrosine, cysteine, serine, threonine, aspartic acid,
arginine and glutamic acid, as well as phosphate
esters of specific amino acids. Indeed, more
progress has been made in the chemistry and
photochemistry of caged polypeptides than
through their application to address important
questions in biology. The present challenge will
be to redress this imbalance by further exploiting the light-directed activation of caged
polypeptides to determine the function and mode
of action of components in signaling, energy
transduction or metabolic processes.
Gerard Marriott and Jeffery W. Walker
Dept of Physiology, University of WisconsinMadison Medical School, 1300 University
Avenue, Madison, WI 53706-1532, USA
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21 Ottl, J. and Marriott, G. (1998) Preparation and
photoactivation of caged fluorophores and caged
proteins using a new class of heterobifunctional,
photocleavable cross-linking reagents,
Bioconjugate Chem. 9, 143–151
22 Arroyo, J.G. et al. (1997) In vivo photoactivation
of caged-thrombin, Thromb. Haemost. 78, 791–793
23 Gillespie, P.G. and Corey, D.P. (1997) Myosin
and adaptation by hair cells, Neuron 19,
955–958
24 Koegler, H., Rueegg, J.C. and Marriott, G. (1998)
Flash photodeprotection of caged myosin: a novel
method to trigger contraction in skinned muscle
fibers, Biophys. J. 74, A335
25 Root, D.D. and Reisler, E. (1992) Cooperativity
of thiol-modified myosin filaments. ATPase and
motility assays of myosin function, Biophys. J.
63, 730–740
26 Mendel, D., Ellman, J.A. and Shultz, P.G. (1991)
Construction of light-activated protein by
unnatural amino acid mutagenesis, J. Am. Chem.
Soc. 113, 2758–2760
27 Miller, J.C. et al. (1998) Flash decaging of tyrosine
sidechains in an ion channel, Neuron 20, 619–624
28 England, P.M. et al. (1997) Site-specific,
photochemical proteolysis applied to ion
channels in vivo, Proc. Natl. Acad. Sci. U. S. A.
94, 11025–11030
update
28 Rathcke, B. (1988) Interactions for pollination
among coflowering shrubs, Ecology 69, 446–457
29 Schürch, S. et al. Effects of ants on the
reproductive success of Euphorbia cyparissias
and associated pathogenic rust fungi, Oikos
(in press)
30 Primack, R.B. (1985) Longevity of individual
flowers, Annu. Rev. Ecol. Syst. 16, 15–38
31 Ashman, T-L. and Schoen, D.J. (1994) How
long should flowers live? Nature 371, 788–791
32 Williamson, G.B. (1982) Plant mimicry:
evolutionary constraints, Biol. J. Linn. Soc. 18, 49–58
33 Waser, N.M. (1983) Competition for pollination
and floral character differences among sympatric
plant species: a review of evidence, in Handbook
of Experimental Pollination Biology (Jones, C.E.
and Little, R.J., eds), pp. 277–292, Van Nostrand
Reinhold
34 Murcia, C. and Feinsinger, P. (1996) Interspecific
pollen loss by hummingbirds visiting flower
mixtures: effects of floral architecture, Ecology
77, 550–560
35 Roy, B.A. and Raguso, R.A. (1997) Olfactory
versus visual cues in a floral mimicry system,
Oecologia 109, 414–426
36 Raguso, R.A. and Roy, B.A. (1998) ‘Floral’ scent
production by Puccinia rust fungi that mimic
flowers, Mol. Ecol. 7, 1127–1136
37 Waser, N.M. (1978) Competition for
hummingbird pollination and sequential
flowering in two Colorado wildflowers, Ecology
59, 934–944
38 Armbruster, W.S. and McGuire, A.D. (1991)
Experimental assessment of reproductive
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interactions between sympatric Aster and
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Constraints and competition in the evolution of
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Thomson, J.D. (1980) Skewed flowering
distributions and pollinator attraction, Ecology
61, 572–579
Dafni, A. et al. (1990) Red bowl-shaped flowers:
convergence for beetle pollination in the
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Smithson, A. and Macnair, M.R. (1997) Negative
frequency-dependent selection by pollinators on
artificial flowers without rewards, Evolution 51,
715–723
Dafni, A. and Calder, D.M. (1987) Pollination by
deceit and floral mimesis in Thelymitra antennifera
(Orchidaceae), Plant Syst. Evol. 158, 11–22
Heinrich, B. (1975) Energetics of pollination,
Annu. Rev. Ecol. Syst. 6, 139–170
Charlesworth, B. (1994) The genetics of adaptation:
lessons from mimicry, Am. Nat. 144, 839–847
Feinsinger, P. et al. (1986) Floral neighborhood
and pollination success in four hummingbirdpollinated cloud forest plant species, Ecology 67,
449–464
Dukas, R. (1987) Foraging behavior of three bee
species in a natural mimicry system: female
flowers which mimic male flowers in Ecballium
elaterium (Curcurbitaceae), Oecologia 74,
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Nilsson, L.A. (1992) Orchid pollination ecology,
Trends Evol. Ecol. 7, 255–259
Caged peptides and proteins:
new probes to study polypeptide
function in complex biological systems
Now that the genomes of Saccharomyces
cerevisiae and Caenorhabditis elegans have
been sequenced, and the sequencing of the
Arabidopsis genome is well under way, cell
biologists are faced with the daunting challenge of establishing the function and mode of
action of thousands of gene products. The ultimate goal of these studies will be to understand how the collective functions of these
proteins and other biomolecules are responsible
for life itself. Among the new tools that need to
be developed to meet these challenges are optical probes and imaging technologies: these
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August 1999, Vol. 4, No. 8
must be capable of identifying and mapping
the interactions and activities of specific proteins
and measuring their associated kinetic parameters with high spatial and temporal resolution.
Light-directed activation of caged (inactive)
compounds should be a valuable technique for
such investigations because it can be used to
manipulate the activity of specific biomolecules in cells (for a comprehensive review of
the methodology involved see Ref. 1). This
technique involves a biologically inert, caged
compound that is specifically and rapidly (ns
to ms) perturbed within a cell using a pulse of
49 Kevan, P.G. (1983) Floral colors through the
insect eye: what they are and what they mean, in
Handbook of Experimental Pollination Biology
(Jones, C.E. and Little, R.J., eds), pp. 3–30,
Van Nostrand Reinhold
50 Chittka, L. et al. (1994) Ultraviolet as a
component of flower reflections, and the colour
perception of hymenoptera, Vision Res. 34,
1489–1508
51 Dafni, A. and Kevan, P.G. (1997) Flower size
and shape: implications in pollination,
Isr. J. Plant Sci. 45, 201–211
52 Dafni, A. (1983) Pollination of Orchis caspia –
a nectarless plant which deceives the pollinators
of nectariferous species from other plant families,
J. Ecol. 71, 467–474
53 Kullenberg, B. (1961) Studies in Ophrys
pollination, Zool. Bidr. Uppsala 34, 1–349
54 Roy, B.A. (1996) A plant pathogen influences
pollinator behavior and may influence
reproduction of non hosts, Ecology 77, 2445–2457
Bitty A. Roy* and Alex Widmer are at the
Geobotanical Institute, Swiss Federal
Institute of Technology (ETH),
Zürichberstr. 38, CH-8044 Zürich,
Switzerland.
*Author for correspondence
(tel 141 01 632 7787;
fax 141 01 632 1215;
e-mail [email protected]).
near ultraviolet light. The effects of a controlled
and local (sub-micron) concentration or activity
jump can then be recorded using timeresolved imaging of a probe of the perturbed
reaction. Specific modulation of protein activity
with light can initiate a specific cell process
that might be used to elucidate the function of
the protein, the kinetics of its activity and the
mechanism of its action (for example, activation
of myosin at the cell cortex and motility).
Over the past 20 years several caged ligands
and substrates have been used to investigate
the molecular basis of intracellular processes,
including muscle contraction2, synaptic transmission3, intracellular signaling4 and motility5.
Unfortunately, small ligands often activate
multiple cellular processes, or can be metabolized to other active species. Caged conjugates
of peptides and proteins are often better suited
for manipulating the activity of a specific protein within a cell6,7. The principle of lightdirected activation of caged polypeptides, and
the potential applications of this technique for
studying the function of specific proteins in
cellular processes are illustrated in Fig. 1.
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(99)01452-1
trends in plant science
update
Functional and spectroscopic
characterization of caged polypeptides
Caged polypeptides should be subjected to a
rigorous biochemical and spectroscopic characterization before and after photoactivation.
These studies should establish the stability of
the caged amino acid residue, the efficiency of
the photoactivation reaction and demonstrate
that photoactivation proceeds without deleterious secondary reactions. It is also important to confirm that the photoproducts of the
photoactivation reaction are benign – this condition has been demonstrated in several applications of light-directed activation of caged
polypeptides in live cells6,8–10.
(a)
Caged peptide Protein
Active peptide
Peptide binding
hu
Caged protein
Ligand
Active protein
Ligand binding
hu
(b)
Cell adhesion protein
Caged peptides
Photolabile reagents were widely used by peptide chemists during the 1970s to protect amino,
phenolic tyrosine and carboxyl groups of amino
acids11. More recently, these reagents have
been used to direct the syntheses of surfacebased, spatially addressable peptide libraries
that can be used to identify novel proteins and
drugs by high throughput screening of chemical and biological libraries12. Peptides specifically caged on arginine10 or tyrosine6,13,
cysteine or thiophosphate groups14 (Fig. 2)
have been described and should prove useful
in studying static or kinetic aspects of specific
protein activity in vivo. A major challenge is
to design a caged peptide that specifically and
potently interferes with the protein or process
under study only after photoactivation.
Caged peptide inhibitors of calmodulin
and myosin light chain kinase
In a comprehensive study involving the development of novel, caged peptides to probe the
function of calmodulin6, the timing and localization of calcium-bound calmodulin (CAM)
activity was assessed in the context of the
motile behavior of eosinophil cells. Several
proteins involved in calcium ion signaling
pathways are activated as a consequence of
CAM binding to specific sequences, including
myosin light chain kinase (MLCK), which
activates the contraction of actomyosin complexes via phosphorylation of myosin light
chain (MLC) (Fig. 3). A 20 amino acid peptide representing the target sequence in
MLCK binds tightly to CAM and inhibits its
signaling function in cell motility. A caged
peptide is designed to act as an inhibitor of
CAM only on removal by light of a protection
group from a single tyrosine residue (caged
RS-20; Fig. 3). The photolabile group is optimized for its rapid photo-release, high quantum yield and the low toxicity of its
photoproduct (Fig. 2). Caged RS-20 binds
CAM 50 times less tightly than the unprotected peptide and is effectively inactive when
assayed in smooth muscle cells known to be
dependent on CAM function. In subsequent
Cell wall
Plasma membrane
Actin and actin-binding
proteins
Signal
transduction
membrane
receptor
Gene
regulation
transcription
factors
Cytoplasm
Cell physiology
Metabolic enzymes
Nucleus
Trends in Plant Science
Fig. 1. Principle of light-directed activation of caged polypeptides. (a) A peptide that inhibits
or activates the activity of a protein is modified with a photolabile group at a specific residue
to block its ability to bind to its target protein. Near ultraviolet irradiation removes the protection
group and generates an active peptide that binds to the target protein. Similarly, a caged protein
can be created by labeling an amino acid in the protein that is essential for its activity (e.g. at
a substrate or ligand-binding site). (b) Photoactivation of microinjected caged polypeptides at
specific cellular locations can be used to investigate the function and kinetics of specific proteins
in processes that might include: membrane receptor signaling, calcium signaling, regulation
of cell adhesion at focal contacts, actin polymerization, cellular energetics or metabolism, and
studies, caged RS-20 has been microinjected
into eosinophil cells and the majority of microinjected cells exhibit a normal motile behavior before exposure to light, but, within
seconds of being irradiated, they round-up and
stop moving because of the effects of the
increased intracellular level of active RS-20.
Irradiated cells resume their motile behavior
at least 50 s after triggering the RS-20
concentration jump, indicating that the
RS-20-mediated inhibition of motility is
reversible. Control experiments show no change
in motile behavior. To demonstrate that the
cessation of motility had been caused by the
inhibition of CAM signaling to MLCK, and
not through other proteins, a caged peptide
inhibitor of MLCK was designed, based on a
peptide sequence in the auto-inhibitory
domain of MLCK (caged LSM-1; Fig. 3).
Functional in vitro assays indicate that caged
LSM-1 inhibits MLCK activity only after
photo-activation. Cells loaded with caged
LSM-1 are fully motile, but again, after photoactivation they round up and stop moving.
Because inhibition of myosin II activity
depends on blocking CAM-MLCK activity
and also on the removal of phosphates by
phosphatase, the timing of the inhibition indicates that phosphate turnover on the light
chain of myosin II must occur with seconds.
These data therefore support the conclusion
that CAM-MLCK-mediated phosphorylation
of myosin II and de-phosphorylation by protein phosphatases are sufficiently rapid to
allow eosinophil cells to respond to the rapid
changes in extracellular gradients of chemoattractant, or fluctuations in intracellular calcium ions (Fig. 3). Because several of the
August 1999, Vol. 4, No. 8
331
trends in plant science
update
(a)
H
CO
H
OCOCl
H
Lysylpolypeptide
NO2
H3CO
H
hu
NH
NO2
NO
+ NH2(CH2)4
H3CO
Tyrosinylpeptide
Br
NO2
OCH3
OH
CO
H
HOOC
CH2 CH
O
HOOC
Fmoctyrosine
CH
+ CO2
OCH3
H
H3COOC
CO
NH
H3CO
OCH3
(b)
O
OCONH (CH2)4 CH
H
O
hu
NH
NO2
NO +
CH2
NH CH CO
(c)
H
CO
H
Br
H
H
Cysteinylpolypeptide
NO2
H3CO
H
CH2 CH
S
NO2
NO
O
CH2
OCH3
Cl
O CO N
+
N(CH3)2
NO2
(f)
NH-(CH2)5CONH-CH2
O
H
N O C
O
H3CO
NH
OCH3
(e)
-
+ HS CH2 CH
H3CO
OCH3
H
CO
hu
NH
H3CO
(d)
O
H3C
H
Biotin
Br
O
NO2
OCH3
O
H
O
NO2
C O N
O
O
Trends in Plant Science
Fig. 2. Chemical strategies used to prepare caged peptides and caged proteins. (a) The e-amino group of lysine can be derivatized with
6-nitroveratryloxycarbonyl chloride to form the corresponding carbamate between a pH value of 8.0 and 9.5 at room temperature. Irradiation of
the carbamate with 365 nm light results in the de-protection of the e-amino group with the concomitant release of carbon dioxide and 3,4dimethoxy-2-nitrosobenzaldehyde. (b) The phenolic oxygen of tyrosine can be protected by alkylation via 2-bromo-a-carboxyl-2-nitrophenyl
methyl ester. Irradiation of the caged peptide rapidly releases the unprotected tyrosine peptide and a relatively non-reactive photoproduct. (c) The
thiol group of a cysteine residue can be protected with 4,5-dimethoxy-2-nitrobenzyl bromide or with 2-bromo-2-(2-nitrophenyl) acetic acid. The
products of photolysis are the free thiol group of cysteine and a potentially thiol-reactive photoproduct. A series of bifunctional photocleavable
reagents have also been used to prepare caged proteins (d and e), and a caged biotin (f).
signaling components in the transduction pathway that leads to eosinophil motility are known
(Fig. 3), and many of these biomolecules can
be caged, it should be possible to use the photoactivation technique to completely describe
the intracellular kinetics of this pathway.
Caged proteins
A caged protein is defined as a protein whose
activity is inhibited following covalent modification of an essential amino acid with a photolabile group – irradiation of a caged protein
with near ultraviolet light removes the protection group from the conjugate and restores its
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August 1999, Vol. 4, No. 8
activity7,15. For example, a caged G-actin conjugate has been described in which lysine
residues essential for actin polymerization
were modified with 6-nitroveratryloxycarbonyl chloride7. Ultraviolet irradiation of
caged actin triggers a photoactivation reaction that liberates native, polymerizationcompetent G-actin, carbon dioxide and
3,4-dimethoxy-2-nitrosobenzaldehyde (Fig. 2).
The same approach has been used to prepare
caged conjugates of profilin16 and the transcription factor GAL418 (Ref. 9). The same
photolabile group attached to lysine residues
of a monoclonal antibody has been used to
prepare a caged antibody that could only bind
its antigen after photoactivation17. A caged
heavy meromyosin (HMM) has also been prepared by labeling an essential cysteine residue
with the photolabile reagent, 4,5-dimethoxy2-nitrobenzenzylbromide15 (Fig. 2), whereas a
related molecule has been used to prepare a
caged pore-forming protein18. In general, cysteine residues are more selectively labeled
compared with lysine because of their lower
frequency in proteins and the higher reactivity of the thiol group at pH 6–8. In addition,
cysteine-mutagenesis can be employed to
design a protein for better caging chemistry
trends in plant science
update
and photoactivation18. Another method to prepare caged proteins is to use photocleavable
crosslinking reagents (Fig. 2). Here, the protein is first modified at a lysine or cysteine
residue using the photocleavable reactive
group of a bifunctional reagent. If the protein
activity is caged after this reaction, then the
second reactive group of the crosslinking
reagent is used to target the caged protein to
another biomolecule, or to a specific location,
via an antibody for later photo-activation19–21.
Photocleavable crosslinking reagents have
been used in this way to prepare caged ricin
for targeting to specific cancer cells19: a fluorescent caged actin monomer21 (Fig. 1) and a
caged actin dimer20 (Fig. 2). More specialized
methods to prepare caged proteins include the
use of photocleavable cinnamate ester substrates to target a reactive serine residue in
thrombin22, or by employing in vitro translation techniques (as discussed here).
Caged myosin
Motor proteins, powered by the hydrolysis of
ATP, are present in all cell types, but the
mechanisms that control the organization and
function of active motor proteins are poorly
understood. Caged motor proteins would be
extremely useful for unravelling spatial and
temporal aspects of motor protein function23.
This approach requires the development of
caged motor proteins that can be incorporated
into such systems, or the development of
methods for modifying endogenous motors with
a caging reagent. Progress towards these goals
has been achieved in studies on light-directed
activation of a caged myosin sub-fragment,
HMM (Refs 15,24). The function of myosin II,
a chemo-mechanical enzyme, is mediated by
coupling two different activities in the motor
molecule. The first, an F-actin-activated Mg2+ATPase, is located in the head region, whereas
the second uses free energy derived from ATP
hydrolysis to drive a stroke of the long a-helix
in the neck that produces a 5 nm translation of
the bound actin filament.
Recent investigations aimed at understanding
the molecular basis of myosin function have
focused on the region around Cys707, which is
close to the fulcrum of the long a-helix. Most
chemical modifications of myosin at this residue
elevate the Mg2+-activated ATPase activity of
the conjugate and inhibit its ability to move actin
filaments (as measured in an in vitro motility
assay sensitive to the chemo-mechanical
coupling25). To elaborate on the importance of the
hinge region in coupling the two activities of
HMM: 4,5-dimethoxy-2-nitrobenzenzylbromide
has been employed to specifically modify
Cys707. As expected, the solution-based Mg2+activated ATPase activity of this conjugate is
higher than native HMM. The functional activity of HMM is determined using a motility assay
on the following surface-bound preparations:
Rho
(a)
MLC
IP3
2+
Ca
CAM
MLCK
MLC
Rho kinase
MBS P
MBS
MLCP
MLCP
MBS
P
Phosphatase?
Actomyosin ATPase
Small GTPase?
(b)
Caged RS-20
+
NH3 -ARRKYQKTGHAVRAIGRLSS-CO2
-
Caged tyrosine
Caged LSM
+
NH3 -LSKDRMKKYMARR-CO2
-
(c)
Net force
Net force
Myosin II
Phosphorylated
myosin II filaments
Actin filaments
Ca2+ CAM
ATP
Pi
Net force
MLCK
ADP
MLCP
Trends in Plant Science
Fig. 3. Essential biomolecules and their sequence in the calcium-mediated signaling pathway
regulating actomyosin contraction during cell motility. (a) Caged biomolecules that could be
used to further define this signaling pathway include calcium ions, inositol triphosphate and
polypeptide inhibitors or activators of Rho kinase, the myosin-binding subunit (MBS) of
myosin light chain phosphatase (MLCP), MLCP and myosin II. (b) Caged RS-20 was prepared by introducing a photolabile tyrosine at position 5 in the myosin light chain kinase
(MLCK) sequence targeted by calmodulin. Photoactivation of the caged peptide generates the
deprotected RS-20, which inhibits calmodulin 50 times more effectively than caged RS-20.
Similarly, caged LSM-1 was prepared by introducing a photolabile tyrosine residue at position nine in a MLCK auto-inhibitory sequence. (c) Schematic of a eukaryotic cell undergoing locomotion. One process thought to be important is the phosphorylation of the myosin
light chain (MLC) of myosin II by Ca2+-calmodulin (CAM)-dependent MLCK. The timing of
this process was examined by rapid, localized photorelease of caged peptides in actively
motile cells6. Cessation of motility owing to inhibition of CAM or MLCK occurred within
10 s, demonstrating that turnover of active phosphorylated myosin II occurs in this time
domain. The organization of active CAM and MLCK can be established with these caged
peptides if peptide diffusion is restricted.
• Native HMM.
• HMM modified on Cys707 with the caged
reagent (caged HMM).
• Caged HMM after irradiation with a pulse
of near ultraviolet light.
Native HMM supports the movement of actin
filaments at a velocity of 3–4 mm/s, whereas
no movement is observed with caged HMM.
HMM is therefore caged in the sense that the
two activities in the conjugate are uncoupled.
However, irradiation with a short pulse of
365 nm light removes the protection group
from Cys707, which results in most actin filaments sliding in the image field at an average
velocity of 2 mm/s. These, and other studies,
suggest that modifying Cys707 with chemical
groups physically hinders the progression of
an ATP hydrolysis-dependent conformational
transition from the head to the neck region that
ordinarily leads to the power stroke of the
lever arm. Light-directed removal of the protection group from Cys707 relieves this physical constraint and restores chemo-mechanical
coupling within HMM with a time constant
of 22 ms (Ref. 15). Preliminary studies have
shown that caged myosin can also be prepared
in functional muscle fibers treated with 4,5dimethoxy-2-nitrobenzenzylbromide. Force
generation is severely impaired in the caged
myosin-containing muscle fibers – yet, as in
August 1999, Vol. 4, No. 8
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trends in plant science
update
the previous study, chemomechanical force
production recovers in the fiber about 20 ms after
removing protection groups with near ultraviolet light24. Further in vivo photoactivation experiments are planned to obtain a better understanding of how the structural dynamics in the
myosin II molecule are related to muscle contraction. This question could be addressed by
recording time-resolved measurements of force
in conjunction with time-resolved X-ray structural analysis before and after photoactivation
of the caged myosin in the muscle fiber. Lightdirected activation of caged non-muscle myosin
isoforms microinjected into motile cells is also
feasible and these studies should prove useful in
defining the kinetics and functional properties
of this protein during motility and cytokinesis.
In vitro translation systems
Recently, methods have been described to prepare caged proteins by supplementing in vitro
translation systems with cRNA containing a
rare or nonsense codon and the complimentary anti-codon tRNA charged with a caged
amino acid. Judicious choice of the site of
incorporation of a caged amino acid can lead
to the expression of a caged protein whose
activity can be triggered with a pulse of near
ultraviolet light26. This strategy has been used
to replace the active site aspartate residue of
lysozyme with caged aspartate26. Caged proteins made by in vitro translation systems are
usually only characterized by activity measurements because of the limited amount of
caged protein synthesized27. A similar
approach has been used to incorporate sitespecific photolabile amino acids into ion channels expressed in Xenopus oocytes28, including
a caged nicotinic acetylcholine receptor27.
Here, oocytes were co-injected with a caged
tyrosine-charged tRNA containing the nonsense anticodon (CUA) and a cRNA of the
a-subunit of nicotinic acetylcholine receptor,
in which a single nonsense codon (UAG)
replaces the tyrosine codon. Full inactivation
of receptor channel activity requires masking
two specific tyrosine residues (one per
a-subunit) at a surface loop of the channel
opening. The channel activity of the membrane-incorporated (caged) receptor complex
is recorded with ms time resolution before
and after irradiation of the egg with ms pulses
of near ultraviolet light. This study demonstrated that a substantial fraction of acetylcholine receptor activity can be rapidly
generated after irradiating the oocyte with
several short pulses of near ultraviolet light27.
This powerful approach extends the applications of light-directed activation of protein
activity to proteins that might be difficult to
purify or to specifically label with a caged
reagent, or for caged proteins that might be
difficult to reconstitute into model or natural
membrane systems.
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August 1999, Vol. 4, No. 8
Summary
Light-directed activation of caged compounds
has emerged as a powerful and general technique
to address the function and mechanisms of protein activity in complex molecular environments. Photoactivation of biomolecules from
ions to second messengers and from metabolites
to polypeptides can be used to reveal information
about the timing and spatial regulation of the
distribution and activity of the biomolecule in
cells and tissues. More recently, reagents and
labeling techniques have been introduced to cage
the activity of almost any biologically active
polypeptide by in vitro or in vivo labeling of
essential amino acids. These include lysine, tyrosine, cysteine, serine, threonine, aspartic acid,
arginine and glutamic acid, as well as phosphate
esters of specific amino acids. Indeed, more
progress has been made in the chemistry and
photochemistry of caged polypeptides than
through their application to address important
questions in biology. The present challenge will
be to redress this imbalance by further exploiting the light-directed activation of caged
polypeptides to determine the function and mode
of action of components in signaling, energy
transduction or metabolic processes.
Gerard Marriott and Jeffery W. Walker
Dept of Physiology, University of WisconsinMadison Medical School, 1300 University
Avenue, Madison, WI 53706-1532, USA
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