 掲載年 |
タイトル |
著者 |
掲載誌 |
概要 |
|
1998 |
Validation of a high throughput screen to detect potential modulators of Ca-2+ release-activated Ca-2+ channels in Jurkat T-cells. |
Bridgland-Taylor,
M. H.; Dale, I. L.; Alexander, P. D.;
Tucker, E.; Midha, A.; Randall, V.
A.; Sullivan, E.; Pollard, C. E. |
British Journal of
Pharmacology, VOL. 125, NO. PROC. SUPPL.1998,
82P page(s) |
DESCRIPTOR(S)- *ANALYTICAL
METHOD; *BIOCHEMISTRY AND BIOPHYSICS;
*CALCIUM ION; *CALCIUM ION CHANNEL;
*CALCIUM RELEASE-ACTIVATED; *CELL BIOLOGY;
*DETECTION METHOD; *EXTRACELLULAR CHELATION;
*FLIPR ASSAY; *FLUORIMETRY; * FLUOROMAX
ASSAY; *HIGH THROUGHPUT; *HUMAN T-CELLS;
*INTRACELLULAR; *JURKAT CELL LINE;
*LOW THROUGHPUT; *MEETING POSTER; *METHODOLOGY;
*MODULATION; *SCREENING METHOD; *THAPSIGARGIN;
*VALIDATION |
|
1998 |
Unfolding of the tetrameric loop deletion mutant of ROP protein is a second-order reaction. |
Lassalle, M. W.; Hinz,
H-J. |
Biochemistry, VOL.
37, NO. 23, 1998, PP. 8465-8472 |
Comprehensive kinetic
studies were carried out on the unfolding
properties of RM6 as a function of
GdnHCl concentration and temperature.
This protein is a mutant resulting
from the dimeric wild-type CoLE1-ROP
protein by deletion of 5 amino acids
(Asp 30, Ala 31, Asp 32, Glu 33, Gln
34) in the loop of each monomer. The
deletion has dramatic consequences.
The dimeric 4-alpha.-helix structure
characteristic of the wild-type protein
is completely reorganized and the RM6
structure can be described as a tetrameric
alpha helix of extended monomers without
loops. These extraordinary structural
changes are accompanied by an enormous
increase in transition temperature
from 71 to 101 degree C. These features
have been discussed in a separate publication
(1). The remarkable change in thermal
stability of RM6 should be reflected
in significant changes in the folding
rate constants. This was observed in
the present unfolding studies. Decay
of tetrameric RM6 was monitored by
circular dichroism (CD) and fluorescence
to probe for changes in both secondary
and tertiary structure, respectively.
The identity of the kinetic parameters
obtained from the two techniques supports
the view that secondary and tertiary
structure break down simultaneously.
However, the most intriguing result
is the finding that unfolding of tetrameric
RM6 can be described very well by a
second-order reaction. The magnitude
of the second-order rate constant k-2
varies dramatically with both temperature
and denaturant concentration. At 25
degree C and 6.5 M GdnHCl concentration
k-2 is 4200 L cntdot (mol of dimer)-1
cntdot s-1, whereas at 4.4 M GdnHCl
a value of k-2= 0.9 L cntdot (mol of
dimer)-1 cntdot s-1 is observed. Correspondingly,
apparent activation enthalpies show
a strong increase from DELTA-H = 29.1
kJ cntdot mol-1 at 6.5 M GdnHCl to
DELTA-H = 79.7 kJ cntdot mol-1 at 4.4
M GdnHCl. A mechanism involving a dimeric
intermediate is suggested which permits
a consistent interpretation of the
findings.
DESCRIPTOR(S)- *AMINO ACIDS; *ANALYSIS;
*ANALYSIS-CHARACTERIZATION TECHNIQUES;
*ANALYTICAL METHOD; *BIOCHEMISTRY
AND BIOPHYSICS; *CIRCULAR DICHROISM;
*EQUIPMENT; *FLUORIMETRY; *MOLECULAR
STRUCTURE; *PROTEIN UNFOLDING MECHANISMS;
*PROTEINS; *RM6 GENE; *ROP PROTEIN;
*SPECTROSCOPIC TECHNIQUES; *SPEX
FLUOROMAX FLUORIMETER; *TEMPERATURE
EFFECTS; *TETRAMERIC LOOP DELETION
MUTANT; *UNFOLDING REACTION
|
|
1998 |
Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination: ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA. |
Gumbs, O. H.; Shaner, S. L. |
Biochemistry, VOL.
37, NO. 33, 1998, PP. 11692-11706 |
The Escherichia coli
RecA protein promotes DNA strand exchange
in homologous recombination and recombinational
DNA repair. Stopped-flow kinetics and
fluorescence resonance energy transfer
(FRET) were used to study RecA-mediated
strand exchange between a 30-bp duplex
DNA and a homologous single-stranded
50mer. In our standard assay, one end
of the dsDNA helix was labeled at apposing
5' and 3' ends with hexachlorofluorescein
and fluorescein, respectively. Strand
exchange was monitored by the increase
in fluorescence emission resulting
upon displacement of the fluorescein-labeled
strand from the initial duplex. The
potential advantages of FRET in study
of strand exchange are that it noninvasively
measures real-time kinetics in the
previously inaccessible millisecond
time regime and offers great sensitivity.
The oligonucleotide substrates model
short-range mechanistic effects that
might occur within a localized region
of the ternary complex formed between
RecA and long DNA molecules during
strand exchange. Reactions in the presence
of ATP with 0.1 mu-M duplex and 0.1-1.0
mu-M ss50mer showed triphasic kinetics
in 600 s time courses, implying the
existence of three mechanistic steps
subsequent to presynaptic filament
formation. The observed rate constants
for the intermediate phase were independent
of the concentration of ss50mer and
most likely characterize a unimolecular
isomerization of the ternary complex.
The observed rate constants for the
first and third phases decreased with
increasing ss50mer concentration. Kinetic
experiments performed with the nonhydrolyzable
analogue ATP-gamma-S showed overall
changes in fluorescence emission identical
to those observed in the presence of
ATP. In addition, the observed rate
constants for the two fastest reaction
phases were identical in ATP or ATP-gamma-S.
The observed rate constant for the
slowest phase showed a 4-fold reduction
in the presence of ATP-gamma-S. Results
in ATP-gamma-S using an alternate fluorophore
labeling pattern suggest a third ternary
intermediate may form prior to ssDNA
product release. The existence of two
or three ternary intermediates in strand
exchange with a 30 bp duplex suggests
the possibility that the step size
for base pair switching may be 10-15
bp. Products of reactions in the presence
of ATP and ATP-gamma-S, with and without
proteinase K treatment, were analyzed
on native polyacrylamide gels. In reactions
in which only short-range Rec-A-DNA
interactions were important, ATP hydrolysis
was not required for recycling of RecA
from both oligonucleotide products.
Hydrolysis or deproteinization was
required for RecA to release the heteroduplex
product, but not the outgoing single
strand.
DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION
TECHNIQUES: CB; *ATP; *BIOCHEMISTRY
AND BIOPHYSICS; *CHARACTERIZATION
METHOD; *CHEMILUMINESCENT DETECTION;
*DETECTION METHOD; *DETECTION-LABELING
TECHNIQUES; *DNA; *EMISSION SPECTRA;
*EQUIPMENT; *FLUORESCENCE RESONANCE
ENERGY TRANSFER TECHNIQUE; *GEL ELECTROPHORESIS;
*GENE MAPPING; *HI-TECH SCIENTIFIC
SF-51 STOPPED FLOW APPARATUS; *HYDROLYSIS;
*METHODOLOGY; *MOLECULAR PROBE TECHNIQUES;
*POLYACRYLAMIDE GEL ELECTROPHORESIS;
*PROTEIN-DNA INTERACTION; *RECA PROTEIN;
*RECOMBINANT DNA TECHNOLOGY; *SEPARATION
METHOD; *SOUTHERN BLOTTING; *SPECTROSCOPIC
TECHNIQUES: CB; *SPEX FLUOROMAX ;
*STOPPED FLOW KINETICS TECHNIQUE
|
|
1998 |
The molecular mechanism of pneumolysin, a virulence factor from Streptococcus pneumoniae. |
Rossjohn, J.; Gilbert,
R. J. C.; Crane, D.; Morgan, P. J.;
Mitchell, T. J.; Rowe, A. J.; Andrew,
P. W.; Paton, J. C.; Tweten, R. K.;
Parker, M. W. |
Journal of Molecular
Biology, VOL. 284, NO. 2, 1998, PP.
449-461 |
Pneumolysin, a member
of the thiol-activated cytolysin family
of toxins, is a virulence factor from
the Gram-positive bacterium Streptococcus
pneumoniae. The toxin forms large oligomeric
pores in cholesterol-containing membranes
of eukaryotic cells. A plethora of
biochemical and mutagenesis data have
been published on pneumolysin, since
its initial characterization in the
1930s. Here we present an homology
model of the monomeric and oligomeric
forms of pneumolysin based on the recently
determined crystal structure of perfringolysin
O and electron microscopy data. A feature
of the model is a striking electronegative
surface on parts of pneumolysin that
may reflect its cytosolic location
in the bacterial cell. The models provide
a molecular basis for understanding
the effects of published mutagenesis
and biochemical modifications on the
toxic activity of pneumolysin. In addition,
spectroscopic data are presented that
shed new light on pneumolysin activity
and have guided us to hypothesise a
detailed model of membrane insertion.
These data show that the environment
of some tryptophan residues changes
on insertion and/or pore formation.
In particular, spectroscopic analysis
of a tryptophan mutant, W433F, suggests
it is the residue mainly responsible
for the observed effects. Furthermore,
there is no change in the secondary
structure content when the toxin inserts
into membranes. Finally, the basis
of the very low activity shown by a
pneumolysin molecule from another strain
of S. pneumoniae may be due to the
movements of a key domain-domain interface.
The molecular basis of pneumolysin-induced
complement activation may be related
to the structural similarity of one
of the domains of pneumolysin to Fc,
rather than the presumed homology of
the toxin to C-reactive protein as
previously suggested.
DESCRIPTOR(S)- *ACTIVITY ASSAYS;
*ANALYSIS; *ANALYTICAL METHOD; *BIOCHEMISTRY
AND BIOPHYSICS; *C-REACTIVE PROTEIN;
*CIRCULAR DICHROISM; *EQUIPMENT;
*FLUORESCENCE SPECTROSCOPY; *HEMOLYTIC
ACTIVITY ASSAY; *JASCO J-720 SPECTROPOLARIMETER;
*METHODOLOGY; *PNEUMOLYSIN; *POLYACRYLAMIDE
GEL ELECTROPHORESIS; *PURIFICATION;
*SDS POLYACRYLAMIDE GEL ELECTROPHORESIS;
*SDS-PAGE; *SPECTROSCOPIC TECHNIQUES:
CB; *SPEX FLUOROMAX SINGLE-PHOTON-COUNTING
SPECTROFLUORIMETER; *STREPTOCOCCUS
PNEUMONIAE; *TOXIN; *VIRULENCE FACTOR
|
|
1998 |
The Cdc42-Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation. |
Rudolph, M. G.; Bayer,
P.; Abo, A.; Kuhlmann, J.; Vetter,
I. R.; Wittinghofer, A. |
Journal of Biological
Chemistry, VOL. 273, NO. 29, 1998,
PP. 18067-18076 |
Wiskott Aldrich syndrome
is a rare hereditary disease that affects
cell morphology and signal transduction
in hematopoietic cells. Different size
fragments of the Wiskott Aldrich syndrome
protein, W4, W7 and W13, were expressed
in Escherichia coli or obtained from
proteolysis. All contain the GTPase
binding domain (GBD), also called Cdc42/Rac
interactive binding region (CRIB),
found in many putative downstream effectors
of Rac and Cdc42. We have developed
assays to measure the binding interaction
between these fragments and Cdc42 employing
fluorescent N-methylanthraniloyl-guanine
nucleotide analogues. The fragments
bind with submicromolar affinities
in a GTP-dependent manner, with the
largest fragment having the highest
affinity, showing that the GBD/CRIB
motif is necessary but not sufficient
for tight binding. Rate constants for
the interaction with W13 have been
determined via surface plasmon resonance,
and the equilibrium dissociation constant
obtained from their ratio agrees with
the value obtained by fluorescence
measurements. Far UV circular dichroism
spectra show significant secondary
structure only for W13, supported by
fluorescence studies using intrinsic
protein fluorescence and quenching
by acrylamide. Proton and 15N NMR measurements
show that the GBD/CRIB motif has no
apparent secondary structure and that
the region C-terminal to the GBD/CRIB
region is alpha-helical. The binding
of Cdc42 induces a structural rearrangement
of residues in the GBD/CRIB motif,
or alternatively, the Wiskott Aldrich
syndrome protein fragments have an
ensemble of conformations, one of which
is stabilized by Cdc42 binding. Thus,
in contrast to Ras effectors, which
have no conserved sequence elements
but a defined domain structure with
ubiquitin topology, Rac/Cdc42 effectors
have a highly conserved binding region
but no defined domain structure in
the absence of the GTP-binding protein.
Deviating from common belief GBD/CRIB
is neither a structural domain nor
sufficient for tight binding as regions
outside this motif are necessary for
structure formation and tight interaction
with Rho/Rac proteins.
DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION
TECHNIQUES; *ANALYTICAL METHOD; *BIACORE
AB; *BIACORE SYSTEM; *BIOCHEMISTRY
AND BIOPHYSICS; *BRUKER; *BRUKER
DRX-500 SPECTROMETER; *CDC42; *CDC42-NUCLEOTIDE
COMPLEX PURIFICATION; *CDC42-RAC
INTERACTIVE BINDING REGION; *CLONING
TECHNIQUES; *CRIB; *ESCHERICHIA COLI;
*EXPRESSION SYSTEM; *FAR UV CIRCULAR
DICHROISM; *FLUORESCENT PROBE; *
FLUOROMAX SPECTROFLUORIMETER; *GENETIC
METHOD; *GTPASE BINDING DOMAIN; *IMAGING
TECHNIQUES; *INTERACTIONS; *ISOLATION-PURIFICATION
TECHNIQUES: CB; *JASCO; *J710 SPECTROPOLARIMETER;
*LABORATORY EQUIPMENT; *METHODOLOGY;
*N-METHYLANTHRANILOYL-GUANINE NUCLEOTIDE
ANALOGUE; *NITROGEN-14 NMR; *PROTON
NMR; *PURIFICATION METHOD; *RHO-RAC
PROTEINS; *SPECTROSCOPIC TECHNIQUES:
CB; *SPEX INDUSTRIES; *STRUCTURE
FORMATION; *SURFACE PLASMON RESONANCE;
*TIGHT BINDING; *WASP FRAGMENT CLONING;
*WASP FRAGMENT PURIFICATION; *WISKOTT
ALDRICH SYNDROME PROTEIN
|
|
1998 |
Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein. |
Raffy, S.; Sassoon,
N.; Hofnung, M.; Betton, J-M. |
Protein Science, VOL.
7, NO. 10, 1998, PP. 2136-2142 |
We previously identified
and characterized amino acid substitutions
in a loop connecting helix I to strand
B, the alpha-I/beta-B loop, of the
N-domain that are critical for in vivo
folding of the maltose-binding protein
(MalE31). The tertiary context-dependence
of this mutation in MalE folding was
assessed by probing the tolerance of
an equivalent alpha-beta loop of the
C-domain to the same amino acid substitutions
(MalE219). Moving the loop mutation
from the N- to the C-domain eliminated
the in vivo misfolding step that led
to the formation of inclusion bodies.
In vitro, both loop variants exhibited
an important decrease of stability,
but their intrinsic tendency to aggregate
was well correlated with their periplasmic
fates in Escherichia coli. Furthermore,
the noncoincidence of the unfolding
and refolding transition curves and
increase of light scattering during
the refolding of MaIE31 indicate that
a competing off-pathway reaction could
occurs on the folding pathway of this
variant. These results strongly support
the notion that the formation of supersecondary
structures of the N-domain is a rate-limiting
step in the folding pathway of MalE.
DESCRIPTOR(S)- *ALPHA-BETA LOOP;
*AMINO-TERMINAL DOMAIN; *ANALYSIS;
*ANALYSIS-CHARACTERIZATION TECHNIQUES:
CB; *ANALYTICAL METHOD; *BIOCHEMISTRY
AND BIOPHYSICS; *ESCHERICHIA COLI;
*EXPRESSION SYSTEM; *EXPRESSION VECTOR;
* FLUOROMAX SPECTROFLUOROMETER; *FOLDING
PATHWAY; *GEL ELECTROPHORESIS; *LABORATORY
EQUIPMENT; *LIGHT-SCATTERING ANALYSIS;
*MALTOSE-BINDING PROTEIN; *METHODOLOGY;
*MISFOLDING SUBSTITUTIONS; *MOLECULAR
GENETIC METHOD; *MUTAGENESIS; *MUTANT;
*PROTEIN ENGINEERING; *P1H PLASMID;
*SDS-PAGE; *SDS-POLYACRYLAMIDE GEL
ELECTROPHORESIS; *SITE-DIRECTED MUTAGENESIS;
*STABILITY; *STRAIN-POP6499; *STRAIN-POP6590;
*STRUCTURE; *WILD-TYPE
|
|
1998 |
Structural transitions accompanying the activation of peptide binding to the endoplasmic reticulum Hsp90 chaperone GRP94. |
Wearsch, P. A.; Voglino,
L.; Nicchitta, C. V. |
Biochemistry, VOL.
37, NO. 16, 1998, PP. 5709-5719 |
GRP94, the endoplasmic
reticulum Hsp90 paralog, binds a diverse
array of peptides, a subset of which
are suitable for assembly onto nascent
MHC class I molecules. At present,
the mechanism, site, and regulation
of peptide binding to GRP94 are unknown.
Using VSVS, the immunodominant peptide
epitope of the vesicular stomatitis
virus, and native, purified GRP94,
we have investigated GRP94-peptide
complex formation. The formation of
stable GRP94-VSV8 complexes was slow;
competition studies demonstrated that
peptide binding to GRP94 was specific.
VSV8 binding to GRP94 was stimulated
2-fold or 4-fold, respectively, following
chemical denaturation/renaturation
or transient heat shock. The activation
of GRP94-peptide binding occurred coincident
with a stable, tertiary conformational
change, as identified by tryptophan
fluorescence and proteolysis studies.
Analysis of GRP94 secondary structure
by circular dichroism spectroscopy
indicated an identical alpha-helical
content for the native, chemically
denatured/renatured, and heat-shocked
forms of GRP94. Through use of the
environment-sensitive fluorophores
acrylodan and Nile Red, it was observed
that the activation of peptide binding
was accompanied by enhanced peptide
and solvent accessibility to a hydrophobic
binding site(s). Peptide binding to
native or activated GRP94 was identical
in the presence or absence of ATP or
ADP. These results are discussed with
respect to a model in which peptide
binding to GRP94 occurs within a hydrophobic
binding pocket whose accessibility
is conformationally regulated in an
adenine nucleotide-independent manner.
DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION
TECHNIQUES; *ANALYTICAL GEL FILTRATION
CHROMATOGRAPHY; *ANALYTICAL METHOD;
*AVIV ASSOCIATES CIRCULAR DICHROISM
SPECTROMETER MODEL 62DS; *BIOCHEMISTRY
AND BIOPHYSICS; *CHROMATOGRAPHIC
TECHNIQUES; *CIRCULAR DICHROISM SPECTROSCOPY;
*ENDOPLASMIC RETICULUM; *EQUIPMENT;
*FLUORESCENCE SPECTROSCOPY; * FLUOROMAX
SPECTROFLUOROMETER; *GRP94; *HSP90
CHAPERONE; *METHODOLOGICAL APPROACH;
*METHODSFINDER; *PEPTIDE BINDING;
*PROTEIN PURIFICATION; *PURIFICATION
METHOD; *SPECTROSCOPIC TECHNIQUES;
*SPEX INDUSTRIES INCORPORATED; *STRUCTURE;
*TSK-GEL G3000 SW COLUMN CHROMATOGRAPH
|
|
1998 |
Rapid assembly of Alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution. |
Friedhoff, P.; Schneider,
A.; Mandelkow, E-M.; Mandelkow, E. |
Biochemistry, VOL.
37, NO. 28, 1998, PP. 10223-10230 |
Alzheimer's disease
is characterized by the progressive
deposition of two types of fibers in
the affected brains, the amyloid fibers
(consisting of the A-beta peptide,
generating the amyloid plaques) and
paired helical filaments (PHFs, made
up of tau protein, forming the neurofibrillary
tangles). While the principles of amyloid
aggregation are known in some detail,
the investigation of PHF assembly has
been hampered by the low efficiency
of tau aggregation, the requirement
of high protein concentrations, and
the lack of suitable detection methods.
Here we report a quantitative assay
system that permits monitoring of the
assembly of PHFs in real time by the
fluorescence of dyes such as thioflavine
S or T. Using this assay, we evaluated
parameters that influence the efficiency
of filament formation. Disulfide-linked
dimers of tau constructs representing
the repeat domain assemble into PHFs
most efficiently, but other tau isoforms
or constructs form bona fide PHFs as
well. The rate of assembly is greatly
enhanced by polyanions such as RNA,
heparin, and notably polyglutamate
which resembles the acidic tail of
tubulin. The assembly is optimal at
pH apprx 6 and low ionic strengths
( lt 50 mM) and increases steeply with
temperatures above 30 degree C, indicating
that it is an entropy-driven process.
DESCRIPTOR(S)- *ALZHEIMER-LIKE PAIRED
HELICAL FILAMENTS; *ANALYSIS; *ANALYTICAL
METHOD; *ASSEMBLY MONITORING; *BIOCHEMISTRY
AND BIOPHYSICS; *ELECTRON MICROSCOPY;
*FLUORESCENCE SPECTROSCOPY; *LABORATORY
EQUIPMENT; *METHODOLOGY; *MICROSCOPY:
CB; *MICROTUBULE-ASSOCIATED PROTEIN
TAU; *RAPID ASSEMBLY IN SOLUTION;
*SPECTROSCOPIC TECHNIQUES: CB; *SPEX
FLUOROMAX INSTRUMENT; *SPEX INSTRUMENTS
SA; *VISUALIZATION METHOD
|
|
1998 |
Protoporphyrin IX Fluorescence Induced in Basal Cell Carcinoma by Oral Aminolevulinic Acid. |
Tope, W., Ross, V., Kollias, N. Martin, A., Gillies, R., and Anderson, R. R.; |
Photochemistry and
Photobiology, Volume 67(2), (1998),
pp. 249 - 255. |
|
|
1998 |
Phage P22 tailspike protein: Removal of head-binding domain unmasks effects of folding mutations on native-state thermal stability. |
Miller, S.; Schuler,
B.; Seckler, R. |
Protein Science, VOL.
7, NO. 10, 1998, PP. 2223-2232 |
A shortened, recombinant
protein comprising residues 109-666
of the tailspike endorhamnosidase of
Salmonella phage P22 was purified from
Escherichia coli and crystallized.
Like the full-length tailspike, the
protein lacking the aminoterminal head-binding
domain is an SDS-resistant, thermostable
trimer. Its fluorescence and circular
dichroism spectra indicate native structure.
Oligosaccharide binding and endoglycosidase
activities of both proteins are identical.
A number of tailspike folding mutants
have been obtained previously in a
genetic approach to protein folding.
Two temperature-sensitive-folding (tsf)
mutations and the four known global
second-site suppressor (su) mutations
were introduced into the shortened
protein and found to reduce or increase
folding yields at high temperature.
The mutational effects on folding yields
and subunit folding kinetics parallel
those observed with the full-length
protein. They mirror the in vivo phenotypes
and are consistent with the substitutions
altering the stability of thermolabile
folding intermediates. Because full-length
and shortened tailspikes aggregate
upon thermal denaturation, and their
denaturant-induced unfolding displays
hysteresis, kinetics of thermal unfolding
were measured to assess the stability
of the native proteins. Unfolding of
the shortened wild-type protein in
the presence of 2% SDS at 71 degree
C occurs at a rate of 9.2 times 10-4
S-1. It reflects the second kinetic
phase of unfolding of the full-length
protein. All six mutations were found
to affect the thermal stability of
the native protein. Both tsf mutations
accelerate thermal unfolding about
10-fold. Two of the su mutations retard
thermal unfolding up to 5-fold, while
the remaining two mutations accelerate
unfolding up to 5-fold. The mutational
effects can be rationalized on the
background of the recently determined
crystal structure of the protein.
DESCRIPTOR(S)- *AMICON; *ANALYSIS;
*ANALYTICAL METHOD; *ANION EXCHANGE
CHROMATOGRAPHY; *AVIV 62A-DS SPECTROPOLARIMETER;
*BACTERIOPHAGE P22; *CARY 1 SPECTROPHOTOMETER;
*CENTRICON 30 MICROCONCENTRATOR;
*CIRCULAR DICHROISM; *CLONING METHOD;
*COLUMN CHROMATOGRAPHY; *CRYSTALLIZATION
TECHNIQUES; *DNA AMPLIFICATION; *ENZYMOLOGY;
*ESCHERICHIA COLI; *EXPRESSION SYSTEM;
*FILTRATION TECHNIQUES; *FLUORESCENCE
SPECTROSCOPY; *FOLDING KINETICS;
*FOLDING MUTATIONS; *GEL ELECTROPHORESIS;
*GEL FILTRATION; *HANGING DROP CRYSTALLIZATION;
*HEAD-BINDING DOMAIN; *JASCO; *JASCO
715 SPECTROPOLARIMETER; *LABORATORY
EQUIPMENT; *METHODOLOGY; *MOLECULAR
GENETIC METHOD; *MUTAGENESIS; *NATIVE
STATE; *PARALLEL BETA HELIX; *PCR;
*PHAGE P22 TAILSPIKE ENDORHAMNOSIDASE;
*POLYMERASE CHAIN REACTION; *PROTEIN
ENGINEERING; *PROTEIN FOLDING INTERMEDIATES;
*PURIFICATION METHOD; *SDS-PAGE;
*SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS;
*SECOND-SITE SUPPRESSOR MUTATIONS;
*SILVER STAINING; *SITE-DIRECTED
MUTAGENESIS; *SPECTROSCOPIC TECHNIQUES:
CB; *SPECTROSCOPY: CB; *SPEX FLUOROMAX
; *STAINING-VISUALIZATION; *STRAIN-JM83;
*THERMA STABILITY; *VARIAN
|
|
1998 |
Monitoring the sizes of denatured ensembles of staphylococcal nuclease proteins: Implications regarding m values, intermediates, and thermodynamics. |
Baskakov, I. V.; Bolen,
D. W. |
Biochemistry, VOL.
37, NO. 51, 1998, PP. 18010-18017 |
Fluorescence and size-exclusion
chromatography (SEC) are used to monitor
urea denaturation of wild-type staphylococcal
nuclease (SN) as well as the m+ and
m- mutants A69T and V66W, respectively.
It is found that the SEC partition
coefficient, 1/K-d, is directly proportional
to the Stokes radii of proteins. From
the Stokes radii, the denatured ensembles
of the three proteins are found to
be highly compact in the limit of low
urea concentration and expand significantly
with increasing urea concentration.
The m values from fluorescence-detected
denaturation of the SN proteins are
generally considered to reflect the
relative sizes of denatured ensembles.
However, the rank order of m values
of the SN proteins studied do not correspond
to the rank order of denatured ensemble
sizes detected by 1/K-d, suggesting
that m values reflect more than just
surface area increases on denaturation.
SEC provides two complementary ways
to demonstrate the existence of intermediates
in urea denaturation and illustrates
that V66W undergoes a three-state transition.
Fluorescence-detected urea denaturations
of A69T and wt SN do not correspond
with 1/K-d-detected denaturation profiles,
a result that would ordinarily mean
that the transitions are non-two-state.
However, this interpretation fails
to recognize the rapidly changing size
and thermodynamic character of the
denatured ensembles of these proteins
both within and outside of the transition
zone. The implications of the changing
sizes and thermodynamic character of
the denatured ensembles for SN proteins
are manifold, requiring a reconsideration
of the thermodynamics of proteins whose
denatured ensembles behave as those
of SN proteins.
DESCRIPTOR(S)- *ANALYSIS; *ANALYSIS-CHARACTERIZATION
TECHNIQUES: CB; *ANALYTICAL METHOD;
*CHROMATOGRAPHIC TECHNIQUES; *DENATURATION;
*ELECTROPHORETIC TECHNIQUES; *ENZYMOLOGY;
*EQUIPMENT; *M VALUES; *MOLECULAR
CHARACTERISTICS; *MOLECULAR INTERMEDIATES;
*MOLECULAR SIZED; *NUCLEASES; *PHENOMENEX;
*PHENOMENEX BIOSEP SEC-S3000 HPLC
SYSTEM; *PROTEIN DENATURATION; *PROTEIN
FOLDING MECHANISMS; *PROTEINS; *SDS-POLYACRYLAMIDE
GEL ELECTROPHORESIS; *SIZE-EXCLUSION
CHROMATOGRAPHY; *SPECTROFLUORIMETRY;
*SPEX; *SPEX FLUOROMAX SPECTROFLUORIMETER;
*STAPHYLOCOCCAL NUCLEASE PROTEINS;
*STAPHYLOCOCCI; *THERMODYNAMICS
|
|
1998 |
Flexibility of helix 2 in the human glutathione transferase P1-1. Time-resolved fluorescence spectroscopy. |
Stella, L.; Caccuri,
A. M.; Rosato, N.; Nicotra, M.; Lo,
Bello M.; De Matteis, F.; Mazzetti,
A. P.; Federici, G.; Ricci, G. |
Journal of Biological
Chemistry, VOL. 273, NO. 36, 1998,
PP. 23267-23273 |
Time-resolved fluorescence
spectroscopy and site-directed mutagenesis
have been used to probe the flexibility
of alpha-helix 2 (residues 35-46) in
the apo structure of the human glutathione
transferase P1-1 (EC 2.5.1.18) as well
as in the binary complex with the natural
substrate glutathione. Trp-38, which
resides on helix 2, has been exploited
as an intrinsic fluorescent probe of
the dynamics of this region. A Trp-28
mutant enzyme was studied in which
the second tryptophan of glutathione
transferase P1-1 is replaced by histidine.
Time-resolved fluorescence data indicate
that, in the absence of glutathione,
the apoenzyme exists in at least two
different families of conformational
states. The first one (38% of the total
population) corresponds to a number
of slightly different conformations
of helix 2, in which Trp-38 resides
in a polar environment showing an average
emission wavelength of 350 nm. The
second one (62% of the total population)
displays an emission centered at 320
nm, thus suggesting a quite apolar
environment near Trp-38. The interconversion
between these two conformations is
much slower than 1 ns. In the presence
of saturating glutathione concentrations,
the equilibrium is shifted toward the
apolar component, which is now 83%
of the total population. The polar
conformers, on the other hand, do not
change their average decay lifetime,
but the distribution becomes wider,
indicating a slightly increased rigidity.
These data suggest a central role of
conformational transitions in the binding
mechanism, and are consistent with
NMR data (Nicotra, M., Paci, M., Sette,
M., Oakley, A. J., Parker, M. W., Lo
Bello, M., Caccuri, A. M., Federici,
G., and Ricci, G. (1998) Biochemistry
37, 3020-3027) and pre-steady state
kinetic experiments (Caccuri, A. M.,
Lo Bello, M., Nuccetelli, M., Nicotra,
M., Rossi, P., Antonini, G., Federici,
G., and Ricci, G. (1998) Biochemistry
37, 3028-3034) indicating the existence
of a pre-complex in which GSH is not
firmly bound to the active site.
DESCRIPTOR(S)- *ANALYSIS; *ANALYTICAL
METHOD; *EC 2.5.1.18; *ENZYMOLOGY;
*ESCHERICHIA COLI; *EXPRESSION SYSTEM;
*HELIX 2 FLEXIBILITY; *HUMAN GLUTATHIONE
TRANSFERASE P1-1; *LABORATORY EQUIPMENT;
*METHODOLOGY; *MOLECULAR GENETIC
METHOD; *MUTAGENESIS; *NEODYMIUM-YAG
LASER; *PROTEIN ENGINEERING; *SITE-DIRECTED
MUTAGENESIS; *SPECTROSCOPY: CB; *SPEX;
*SPEX FLUOROMAX PHOTON COUNTING SPECTROFLUOROMETER;
*TIME-RESOLVED FLUORESCENCE SPECTROSCOPY
|
|
1998 |
Definition of the specific roles of lysolecithin and palmitic acid in altering the susceptibility of dipalmitolyphosphatidylcholine bilayers to phospholipase A-2. |
Henshaw, J. B.; Olsen,
C. A.; Farnbach, A. R.; Nielson, K.
H.; Bell, J. D. |
Biochemistry, VOL.
37, NO. 30, 1998, PP. 10709-10721 |
Bilayers composed
of phosphatidylcholine initially resist
catalysis by phospholipase A-2. However,
after a latency period, they become
susceptible when sufficient reaction
products (lysolecithin and fatty acid)
accumulate in the membrane. Temperature
near the main bilayer phase transition
and calcium concentration modulate
the effectiveness of the reaction products.
The purpose of this study was to examine
the individual contributions of lysolecithin
and palmitic acid to the susceptibility
of dipalmitoylphosphatidylcholine vesicles
and to rationalize the effects of temperature
and calcium. Various fluorescent probes
(Prodan, Laurdan, pyrene-labeled fatty
acid, and dansyl-labeled phospholipid)
were used to assess changes in the
ability of the reaction products to
perturb the bilayer and to affect the
interactions with the enzyme. Un-ionized
palmitic acid decreased bilayer polarity
and perturbed the membrane surface
exposing some of the Prodan to bulk
water. Lysolecithin increased bilayer
polarity and the rate of dipolar relaxation
in response to the excited states of
Laurdan and Prodan. A combination of
the individual contributions of each
product was observed when palmitic
acid and lysolecithin were present
together at low calcium, and the effects
of lysolecithin dominated at high calcium.
Palmitic acid, but not lysolecithin,
promoted the binding of phospholipase
A-2 to the bilayer surface in the absence
of calcium. Lysolecithin reduced the
ability of fatty acid to enhance binding
apparently by altering the structure
of fatty acid domains in the membrane.
Furthermore, increased temperature
and ionization of the fatty acid tended
to cause segregation of bound phospholipase
A-2 into domains poor in phospholipid
content which presumably impeded bilayer
hydrolysis. In contrast, unionized
palmitic acid and lysolecithin promoted
hydrolysis by augmenting a step distal
to the adsorption of enzyme to the
bilayer. This kinetic response to lysolecithin
was calcium-dependent. A model accounting
for these varied influences of the
reaction products is presented.
DESCRIPTOR(S)- *AGKISTRODON-PISCIVORUS-PISCIVORUS
VENOM PHOSPHOLIPASE A-2; *ANALYSIS;
*ANALYTICAL METHOD; *AVANTI POLAR
LIPIDS; *BIOCHEMISTRY AND BIOPHYSICS;
*CATALYTIC SUSCEPTIBILITY; *DIPALMITOLYPHOSPHATIDYLCHOLINE
BILAYERS; * FLUOROMAX SPECTROFLUOROMETER;
*GREG PC PHOTON-COUNTING SPECTROFLUOROMETER;
*ISS; *LABORATORY EQUIPMENT; *LYSOLECITHIN;
*METHODOLOGY; *PALMITIC ACID; *PC-1
PHOTON-COUNTING SPECTROFLUOROMETER;
*SIGMA; *SPECTROFLUORIMETRY; *SPECTROSCOPIC
TECHNIQUES: CB; *SPEX INDUSTRIES
|
|
1998 |
Correlation of fluorescence and circular dichroism spectroscopy with electrospray ionization mass spectrometry in the determination of tertiary conformational changes in calcium-binding proteins. |
Veenstra, T. D.; Johnson,
K. L.; Tomlinson, A. J.; Kumar, R.;
Naylor, S. |
Rapid Communications
in Mass Spectrometry, VOL. 12, NO.
10, 1998, PP. 613-619 |
We compared changes
in the fluorescence, circular dichroism
(CD) and multiply charged electrospray
ionization mass spectrometry (ESI-MS)
spectra of three calcium (Ca-2+)-binding
proteins upon the binding of Ca+. The
proteins used were rat brain calbindin
D-28K and two deletion mutants, one
lacking EF-hand 2 (calbindin DELTA-2)
and the other lacking EF-hands 2 and
6 (calbindin DELTA-2,6). Large changes
in the intrinsic protein fluorescence
spectrum were seen upon the addition
of Ca-2+ to calbindin D-28K and DELTA-2,
while a less significant change was
observed for calbindin DELTA-2,6. In
a fluorescent study in which p-toluidinyl-2-naphthalene-6-sulfonate,
a fluorescent probe which binds to
hydrophobic surfaces within proteins,
was used; calbindin D-28K and DELTA-2
again showed a greater change in fluorescence
intensity upon Ca-2+-binding than calbindin
DELTA-2,6. Near UV-CD studies, which
measure changes within the tertiary
structure of a protein, showed greater
changes in the spectrum of calbindin
D-28K and DELTA-2 compared to calbindin
DELTA-2,6 upon Ca-2+binding. Far UV-CD
studies, which measures changes within
the secondary structure of a protein,
however, showed that the spectrum of
all three proteins underwent only minor
changes upon metal-binding. The ESIMS
studies showed that as the proteins
were titrated with Ca-2+ a gradual
shift in the mass envelope from higher
to lower charge states occurs. In the
case of calbindin D-28K and calbindin
DELTA-2, however, a complete shift
in the mass envelope towards the lower
charge states is observed upon saturation
with Ca-2+, whereas for calbindin DELTA-2,6,
the shift in the charge states is still
relatively evenly distributed between
high and low charge states. Changes
within the ESI-MS spectrum observed
upon the addition of Ca-2+ correlated
with Ca-2+-induced changes observed
with near-ultraviolet CD, intrinsic
fluorescence spectroscopy, and spectroscopy
using the fluorescent probe. Changes
in the far ultraviolet-CD spectra of
the calbindins, however, did not correlate
with changes in the ESI-MS spectra
upon calcium binding. The results show
that ESI-MS can be use to detect changes
in the tertiary structure of calcium-binding
proteins induced by the binding of
metal to the proteins.
DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION
TECHNIQUES; *BIOCHEMISTRY AND BIOPHYSICS;
*CALBINDIN; *CALCIUM-BINDING PROTEINS;
*CHARACTERIZATION; *CHARACTERIZATION
METHOD; *CIRCULAR DICHROISM SPECTROSCOPY;
*ELECTROSPRAY IONIZATION MASS SPECTROMETRY;
*EQUIPMENT; *FINNIGAN MAT 900 MASS
SPECTROMETER; *FLUORESCENCE SPECTROSCOPY;
* FLUOROMAX FLUORESCENCE SPECTROMETER;
*JASCO J-710 SPECTROPOLARIMETER;
*METHODOLOGY; *METHODSFINDER; *SPECTROSCOPIC
TECHNIQUES; *SPEX INSTRUMENTS; *TERTIARY
CONFORMATIONAL CHANGES
|
|
1998 |
Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE. |
Pierpaoli, E. V.,
Sandmeier, E., Schoenfeld, H-J., and
Christen, P.; |
Journal of Biological
Chemistry, Volume 273, Number 12, (1998),
pp. 6643 - 6649. |
|
|
1998 |
Calcium-dependent membrane penetration is a hallmark of the C2 domain of cytosolic phospholipase A2 whereas the C2A domain of synaptotagmin binds membranes electrostatically. |
Davletov, B.; Perisic,
O.; Williams, R. L. |
Journal of Biological
Chemistry, VOL. 273, NO. 30, 1998,
PP. 19093-19096 |
C2 domains have been
identified in a wide range of intracellular
proteins, including lipid modifying
enzymes, protein kinase, GTPases, and
proteins involved in membrane trafficking.
Many C2 domains bind membranes in a
calcium-dependent manner. The first
C2 domain from synaptotagmin I (SytIC2A)
and the C2 domain from cytosolic phospholipase
A2 (cPLA2C2) are among the best characterized
C2 domains in terms of their structures
and calcium binding. Here we demonstrate
that the protein lipid interaction
is dramatically different for these
two domains. Photolabeling with 3-(trifluoromethyl)-3-(m-(125I)iodophenyl)diazirine
((125I)TID) in the presence of phospholipid
vesicles indicates that cPLA2C2 penetrates
into the hydrophobic region of the
membrane. Hydrophobic surfaces on cPLA2C2
are exposed even in the absence of
calcium, but only in its presence does
the domain penetrate into the nonpolar
core of the membrane. The interaction
of SytIC2A with phospholipid membranes
is primarily electrostatic with binding
being abolished in 500 mM NaCl. Because
soluble phospholipid head group analogues
do not compete with binding of either
SytIC2A or cPLA2C2 to vesicles, it
is likely that membrane binding by
these domains involves multiple interactions.
DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION
TECHNIQUES: CB; *ANALYTICAL METHOD;
*CALCIUM-DEPENDENT MEMBRANE PENETRATION;
*CYTOSOLIC PHOSPHOLIPASE A2; *C2
DOMAIN; *C2A DOMAIN; *DETECTION-LABELING
TECHNIQUES; *ELECTROSTATIC MEMBRANE
BINDING; *ENZYMOLOGY; *EXPRESSION;
*FLUORESCENCE ANALYSIS; * FLUOROMAX-2
FLUORIMETER; *GEL ELECTROPHORESIS;
*HYDROPHOBIC SURFACES; *ISOLATION-PURIFICATION
TECHNIQUES: CB; *JOBIN YVON-SPEX;
*LABORATORY EQUIPMENT; *LARGE MULTILAMELLAR
VESICLES; *MEMBRANES; *METHODOLOGY;
*MOLECULAR PROBES; *PHASE SEPARATION;
*PROTEIN RADIOLABELING; *PROTEIN-LIPID
INTERACTION; *PURIFICATION; *PURIFICATION
METHOD; *SDS-PAGE; *SDS-POLYACRYLAMIDE
GEL ELECTROPHORESIS; *SEPARATION
METHOD; *SIGMA; *SYNAPTOTAGMIN; *TRITON
X-114; *4,4'-DIANILINO-1,1'-BINAPHTHYL-5,5'-DISULFONIC
ACID
|
|
1998 |
Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. |
Chinchilla, D.; Schwarz,
F. P.; Eisenstein, E. |
Journal of Biological
Chemistry, VOL. 273, NO. 36, 1998,
PP. 23219-23224 |
Shifts in the sigmoidal
kinetics of allosteric threonine deaminase
promoted by isoleucine and valine binding
control branched chain amino acid biosynthesis
in Escherichia coli. A highly conserved
alpha-helix in the C-terminal regulatory
domain of the tetrameric enzyme was
previously implicated in effector binding
and feedback inhibition. Double (447,
451) and triple (447, 451, 454) alanine
replacements for the conserved amino
acids leucine 447, leucine 451, and
leucine 454 in this region yield enzyme
variants that show increased sigmoidality
in steady-state kinetics, and which
are less sensitive to the allosteric
modifiers isoleucine and valine. Equilibrium
binding studies using fluorescence,
enzyme kinetic, and calorimetric approaches
indicate that the enzyme variants possess
reduced affinity for isoleucine and
valine, and suggest that heterotropic
ligands can bind to the same site to
promote their different effects. The
increase in sigmoidal kinetics for
the mutants relative to wild-type threonine
deaminase may be attributable to the
elimination of L-threonine binding
to the effector sites, which activates
the wild-type enzyme. Enzyme kinetic
data and isotherms for active site
ligand binding to the mutants can be
analyzed in terms of a simple two-state
model to yield values for allosteric
parameters that are consistent with
previous estimates based on an expanded
two-state model for homotropic cooperativity
for threonine deaminase.
DESCRIPTOR(S)- *ACTIVE SITE LIGAND
BINDING; *ALLOSTERIC EFFECTOR BINDING;
*AMINO ACID SUBSTITUTIONS; *ANALYSIS;
*ANALYSIS-CHARACTERIZATION TECHNIQUES:
CB; *ANALYTICAL METHOD; *BECKMAN
OPTIMA XL-A ANALYTICAL ULTRACENTRIFUGE;
*CARBOXY-TERMINAL REGULATORY DOMAIN;
*ENZYMOLOGY; *ESCHERICHIA COLI; *FLUORESCENCE
SPECTROSCOPY; * FLUOROMAX 2000 SPECTROFLUORIMETER;
*HOMOTROPIC COOPERATIVITY; *LABORATORY
EQUIPMENT; *METHODOLOGY; *MICROCAL
OMEGA TITRATION CALORIMETER; *MOLECULAR
GENETIC METHOD; *MUTAGENESIS; *MUTANT;
*MUTATION; *PROTEIN ENGINEERING;
*SEDIMENTATION EQUILIBRIUM ANALYSIS;
*SITE-DIRECTED MUTAGENESIS; *SPECTROSCOPIC
TECHNIQUES: CB; *SPEX INDUSTRIES;
*STEADY STATE KINETICS; *THREONINE
DEAMINASE; *TITRATION CALORIMETRY
|
|
1998 |
A method for studying plasma membrane transport with intact cells using computerized fluorometry. |
Wielinga, P. R.; Heijn,
M.; Westerhoff, H.; Lankelma, J. |
Analytical Biochemistry,
VOL. 263, NO. 2, 1998, PP. 221-231 |
A new method is presented
for measuring rapid efflux of fluorescent
compounds from monolayer cells. Cells
grown on a glass coverslip were loaded
with a fluorescent substrate. Thereafter,
the coverslip was installed outside
the light path in a stirred and thermostated
cuvette of a fluorometer. The efflux
was recorded by measuring the changes
of fluorescence in the extracellular
medium. The method was used to study
the kinetics of active and passive
plasma membrane transport of the P-glycoprotein
substrates rhodamine 123 and daunorubicin.
The method has advantages over other
methods: (1) no radioactively labeled
substrate is needed, (2) fluorescence
of the transported substrate is not
compromised by the cells, (3) changes
in the extracellular concentration
of the substrate can be monitored continuously
and therefore a substantial improvement
of the kinetic resolution is obtained,
and (4) the measurement setup is relatively
simple and a standard fluorometer can
be used. From the efflux data, cellular
transport parameters could be calculated,
such as passive permeation coefficients
and active transport rates.
DESCRIPTOR(S)- *ANALYTICAL METHOD;
*COMPUTERIZED FLUOROMETRY; *DAUNORUBICIN;
* FLUOROMAX SPECTROFLUOROMETER; *INTACT
CELLS; *KB-V1 CELL LINE; *KB-3-1
CELL LINE; *LABORATORY EQUIPMENT;
*MEMBRANES; *METHODOLOGY; *P-GLYCOPROTEIN
SUBSTRATE; *PHOTOMETRY: CT; *PLASMA
MEMBRANE TRANSPORT; *RHODAMINE 123;
*SIGMA; *SPECIA; *SPEX INDUSTRIES;
*TRANSPORT
|
|
1998 |
2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: Metal-ion and base sequence effects. |
Stivers, J. T. |
Nucleic Acids Research,
VOL. 26, NO. 16, 1998, PP. 3837-3844 |
Metal-ion and sequence
dependent changes in the stacking interactions
of bases surrounding abasic (AB) sites
in 10 different DNA duplexes were examined
by incorporating the fluorescent nucleotide
probe 2-aminopurine (2-AP), opposite
to the site (AB-AP-opp) or adjacent
to the site (AB-Ap-adj) on either strand.
A detailed study of the fluorescence
emission and excitation spectra of
these AB duplexes and their corresponding
parent duplexes indicates that AB-AP-opp
is significantly less stacked than
2-AP in the corresponding normal duplex.
In general, AB-Ap-adj on the AB strand
is stacked, but AB-Ap-adj on the opposite
strand shows destabilized stacking
interactions. The results also indicate
that divalent cation binding to the
AB duplexes contributes to destabilizaton
of the base stacking interactions of
AB-AP-opp, but has little or no effect
on the stacking interactions of AB-AP-adj.
Consistent with these results, the
fluorescence of AB-AP-opp is 18-30-fold
more sensitive to an externally added
quenching agent than the parent normal
duplex. When uracil DNA glycosylase
binds to AB-AP-opp in the presence
of 2.5 mM MgCl-2, a 3-fold decrease
in fluorescence is observed (K-d =
400 +- 90 nM) indicating that the unstacked
2-AP-opp becomes more stacked upon
binding. On the basis of these fluorescence
studies a model for the local base
stacking interactions at these AB sites
is proposed.
DESCRIPTOR(S)- *ANALYSIS-CHARACTERIZATION
TECHNIQUES: CB; *ANALYTICAL METHOD;
*APPLIED BIOSYSTEMS; *APPLIED BIOSYSTEMS
390 SYNTHESIZER; *BASE SEQUENCE EFFECTS;
*BASE STACKING INTERACTIONS; *CONTINUOUS
FLUORESCENCE KINETIC ASSAY; *DETECTION
METHOD; *DETECTION-LABELING TECHNIQUES;
*DNA; *ESCHERICHIA COLI; *FLUORESCENCE
EMISSION SPECTROSCOPY; *FLUORESCENT
NUCLEOTIDE PROBE; *FLUOROPHOTOMETRY:
CB; *GEL ELECTROPHORESIS; *LABORATORY
EQUIPMENT; *LIQUID CHROMATOGRAPHY;
*METAL-ION EFFECTS; *METHODOLOGY;
*MOLECULAR GENETICS; *OLIGODEOXYNUCLEOTIDE
SYNTHESIS; *POLYACRYLAMIDE GEL ELECTROPHORESIS;
*PURIFICATION METHOD; *REVERSE-PHASE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY;
*REVERSE-PHASE HPLC; *SPEX FLUOROMAX
SPECTROFLUOROMETER; *SYNTHESIS-MODIFICATION
TECHNIQUES; *SYNTHETIC METHOD; *TITRATION;
*URACIL DNA GLYCOSYLASE; *2-AMINOPURINE;
*2-AMINOPURINE FLUORESCENCE STUDIES
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