Transcript 2009/05/21 Lecture

Circular
Dichroism
Spectroscopy
(CD)
Optically active sample vs. polarized light
Incident
linearly
polarized
light.
Resolution of linearly polarized
light into individual right-hand
and left-hand circularly polarized
components.
Elliptically polarized
light produced by
passing the incident
light through an
optically active
sample.
Effect of an optically active sample on the two
circularly polarized components. The sum of
measurements made with these two separate
components must be identical to the result
obtained in part b.
Beer-Lambert Law
A= e x b x c
DA = (eL-eR) × c × l
De = eL-eR
differential absorbance of a 1 mol/l solution in a 1 cm cell
Measured q , ellipticity, is the rotation in degrees
of a 1 dmol/cm3 solution and a pathlength of 1 cm
Mean residue ellipiticity: [q] = q222*MMRW/10*l*c
degrees cm2 dmol-1 residue -1
MMRW: mean residue weight (MW/ amino acid residue number)
l: cell path in cm
c: protein concentration in mg/ml
q x 100 x Mr
Molar ellipticity: [q] =
degree cm2 dmol-1
De = [q] /3298
cx l
c: mg/ml
l: cm
Litre mol-1 cm-1 or Litre (mol residue)-1 cm-1
Applications of CD Spectroscopy
• determining whether a protein is folded, and if
so characterizing its secondary structure, tertiary
structure
• comparing the structures of a protein obtained
from different sources or comparing structures
for different mutants of the same protein
• studying the conformational stability of a protein
under stress -- thermal stability, pH stability, and
stability against denaturants
• determining whether protein-protein interactions
alter the conformation of protein.
CD Spectrum of Protein
Near-UV CD spectrum:
250~350 nm – protein tertiary structure
Far-UV CD spectrum:
190~250 nm – protein secondary structure
※
The signal strength in the near-UV CD region is
much weaker than that in the far-UV CD region.
Far-UV CD spectra
Near-UV CD spectrum
Biochimica et Biophysica Acta 1751, 119 – 139 (2005)
CD spectra of HIV-1 gp41 ectodomain
(A) Far-UV spectra
(B) Near-UV spectra
Electrophoresis 2008, 29, 3175–3182
The influence of detergents on secondary and tertiary structures of
the gp41 ectodomain.
Protein: 10 mM; Solvent:
H2O, 1% PFO, or 1% SDS
In SDS micellar suspension both secondary and tertiary structures
are destabilized, while PFO leaves both structures largely intact.
Fluorescence
Spectroscopy
Excitation and emission of fluorescence
Fluorescence spectroscopy is primarily concerned with
electronic and vibrational states.
Note that non-radiative transitions
relax S2 to S1 much faster than
any of the de-excitation
processes can return S1 to the
ground state (S0).
Schematic of a fluorometer
A fluorometer with a 90° geometry
utilizing a Xe light source
Fluorescence Spectroscopy
• Tryptophan
• Bis-ANS (4,4'-dianilino-1,1'-binaphthyl- 5,5'-disulfonic acid,
dipotassium salt)
• Rhodamine
• NBD (4-chloro-7-nitrobenz-2-oxa-1,3-diazole)
• Tb3+/DPA (dipicolinic acid) leakage experiments
• FRET (Fluorescence resonance energy transfer)
Label on peptide
1. NBD-Rhodamine pair
2. Pyrene-NBD pair
Label on lipid
• Quenching
Tryptophan quenching with Acrylamide
NBD quenching with Co2+
Tryptophan fluorescence
Tryptophan is an important intrinsic fluorescent probe (amino acid),
which can be used to estimate the nature of microenvironment of
the tryptophan. When performing experiments with denaturants,
surfactants or other amphiphilic molecules, the microenvironment of
the tryptophan might change.
For example, if a protein containing a single tryptophan in its 'hydrophobic'
core is denatured with increasing temperature, a red-shift emission
spectrum will appear. This is due to the exposure of the tryptophan to an
aqueous environment as opposed to a hydrophobic protein interior. In
contrast, the addition of a surfactant to a protein which contains a
tryptophan which is exposed to the aqueous solvent will cause a blue
shifted emission spectrum if the tryptophan is embedded in the surfactant
vesicle or micelle.
Proteins that lack tryptophan may be coupled to a fluorophore.
At 295 nm, the tryptophan emission spectrum is dominant over the weaker
tyrosine and phenylalanine fluorescence.
Low-pH-induced conformation change of HA2
38
55
146
|
153
105
76
129
113
Low pH
Nature 371, 37-43 (1994)
Trp fluorescence of HA2 TMD peptide
Ex l: 280 nm
BMC Biology 2008, 6:2
Trp in a polar environment shows a fluorescent maximum at around 350
nm. A shift of emission maximum from 345 to 337 nm and the enhancement of
emission as the TMD peptide in aqueous buffer was added to the vesicular
dispersion indicate the immersion of the TMD peptide in the lipid bilayer.
Bis-ANS fluorescence
Bis-ANS
4,4'-dianilino-1,1'-binaphthyl- 5,5'-disulfonic acid, dipotassium salt
• Bis-ANS binds to the hydrophobic clefts of proteins and exhibits a
significant enhancement of fluorescence upon binding.
• Bis-ANS can be used to investigate structural changes in tubulin
monomers and dimers during time- and temperature-dependent
decay. The bis-ANS binding site on tubulin lies near the contact
region that is critical for microtubule assembly, but it is distinct
from the binding sites for the antimitotic drugs colchicine,
vinblastine, podophyllotoxin and maytansine.
Binding of Bis-ANS to HA2(21-174)
pH 5.0, 37 o C
pH 7.4, 37 o C
4000
3500
3500
3000
3000
2500
Bis-ANS
2000
HA2/Bis-ANS
1500
1000
Fluorescence Intensity
Fluorescence Intensity
Ex l: 290 nm
4000
491 nm
2500
490 nm
Bis-ANS
2000
HA2/Bis-ANS
1500
1000
500
500
518 nm
0
518 nm
0
400
500
600
Wavelength (nm)
700
400
500
600
700
Wavelength (nm)
The increase of bis-ANS fluorescence in the presence of HA2(21-174) in PB buffer solutions was
accompanied by a blue shift of the wavelength of maximal fluorescence. These results were
associated with the exposure of the hydrophobic binding sites of HA2(21-174). The low-pH
induced conformational change caused the higher fluorescence intensity at pH 5 than that at pH
7.4.
Rhodamine fluorescence
Octadecyl rhodamine B (R18)
R18 is a lipophilic cation that has been extensively used as a membrane
probe. Viral particles that have been labeled with high concentrations of
R18 have fluorescence that is highly self-quenched; fusion of the particle
with cell membranes relieves the quenching, making the receptor cell
highly fluorescent.
5(6)-TAMRA
5-(and-6)-carboxytetramethylrhodamine
Tetramethylrhodamine (TMR) is an important fluorophore for preparing
protein conjugates. Under the name TAMRA, the carboxylic acid of TMR
has also achieved prominence as a dye for oligonucleotide labeling and
automated DNA sequencing applications. 5-(and-6)carboxytetramethylrhodamine, succinimidyl ester, is the amine-reactive,
mixed isomer form of TAMRA.
R18 dequenching of HA2 fusion peptide
(A) Kinetics of HA2 FP-induced
dequenching of R18 at different pH
values. The pH of curves a–f are
indicated in
panel B. (B) Wild type fusion peptide
induced percent dequenching as a
function of pH.
%dequenching 
( Ft  Fo )
( F  Fo )
where Ft and F0 are fluorescence intensities
at a given time t and at time zero,
respectively, while F is the fluorescence after
introduction of Triton X-100 and is taken as
fluorescence at infinite dilution of the probe.
Ex l: 530 nm
Chem. Phys. Lipids 2000, 107, 99–106
Dequenching of rhodamine labeled HA2 FP
Self-association of the 25-mer
fusion peptide analogs of HA2 in
DMPC:DMPG (4:1) vesicles at pH
5.0 and 37 C as probed by
fluorescence self-quenching of
rhodamine attached to the N
terminus of the peptides. All four
vesicle-associated labeled peptides
in the membrane-bound state
exhibited moderate dequenching
when solubilized by Triton X-100
indicating a loose oligomerization
for these four peptides tested.
Compact packing or aggregation of
all the peptides tested is
manifested by a highly quenched
rhodamine fluorescence in
aqueous buffer solution.
Biochim. Biophys. Acta 2003, 1612, 41– 51
Rhodamine composition experiments
x=[Rho-peptide]/[Total peptide]
Rhodamine composition experiments detect tight self-association of HA2 TMD and
non-random interaction of TMD:FP association.
The intra-trimeric interaction is detected for x values near 1 since nearly all peptide molecules are
labeled and, therefore, quenching arises predominantly from the close neighbors within the same
trimer. In contrast, for low x values, the probability of finding a pair of labeled peptides is slim and
hence quenching arises mainly from labeled peptides in nearby trimers.
The large self-quenching (i.e. low intensity) of Rhodamine is virtually unchanged in the x = 0.3–1.0
region as the labeled TMD manifests packing of TMD molecules into a tight subunit in the membrane
at pH 5.0 and 7.4. In contrast, labeled FP exhibits less self-quenching, indicative of a loose
association for the peptide molecules.
BMC Biology 2008, 6:2
NBD fluorescence
NBD
4-chloro-7-nitrobenz-2-oxa-1,3-diazole
The nitrobenzoxadiazole (NBD) fluorophore is highly environment-sensitive.
Although it is moderately fluorescent in aprotic solvents, in aqueous solvents it is
almost non-fluorescent. NBD chloride was first introduced in 1968 as a fluorogenic
derivatization reagent for amines. NBD fluoride usually yields the same products
as NBD chloride but is much more reactive.
The fluorophore is moderately polar and its fatty acid analogs and the
phospholipids derived from these probes tend to sense the lipid–water interface
region of membranes instead of the hydrophobic interior. NBD fatty acids are not
well metabolized by living cells. The environmental sensitivity of NBD fatty acids
can be usefully exploited to probe the ligand binding sites of fatty acid and sterol
carrier proteins.
NBD fluorescent spectra of ASLV-IFP
Lipid binding of NBD-IFP (internal
fusion peptide) of the avian sarcoma
and leucosis virus (ASLV) envelope
glycoprotein at pH 7.4 and 37 C.
Increased intensity and the blue-shift
of fluorescence of NBD attached to
the N-terminus of the peptide indicate
embedding of the peptide in the
apolar milieu of membrane bilayer. As
a control, proteinase K digestion of
the peptide disrupts membrane
binding releasing bound NBD and
thus reduces fluorescence of the
fluorophore.
Ex l: 467 nm
Eur. J. Biochem. 2004, 271, 4725–4736
Tb3+/DPA leakage experiments
The method is based on the enhancement of the
lanthanide metal Tb3+ fluorescence when the aromatic
chelator DPA is liganded to the ion.
Tb3+ is encapsulated in the large unilamellar vesicles (LUVs).
The Tb3+-encapsulated liposomes mixed with each of the
tested peptides is used to monitor the leakage activity. The
enhancement of Terbium fluorescence by the external DPA
is due to energy transfer from the aromatic ring of DPA.
Tb3+/DPA leakage experiments
Membrane leakage experiments
using Tb3+/DPA assay to
monitor membrane activity of
TMD, FP and TMD:FP complex
of HA2. Both FP and FP:TMD
display dose-dependent
leakage activity whereas TMD
alone exhibits little activity. It is
noted that the characteristic
time of leakage is approximately
200 s for P/L = 0.05.
Ex l: 270 nm
Em l: 490nm
BMC Biology 2008, 6:2
FRET (Fluorescence Resonance Energy Transfer)
A donor chromophore in its excited state can transfer energy by
a non-radiative, long-range dipole-dipole coupling
mechanism to an acceptor chromophore in close proximity
(typically <10 nm). This energy transfer mechanism is termed
Förster resonance energy transfer and when both molecules
are fluorescent, the term "fluorescence resonance energy
transfer" is often used.
The Förster distance (R0)
at which the FRET efficiency is 50%
• R0 of NBD-Rho pair (donor-acceptor) is about 56 Å
NBD
Ex 470 nm
Em 530 nm
Rhodamine Ex 530 nm
Em 580 nm
• R0 of Pyrene-NBD pair (donor-acceptor) is about 33 Å
Pyrene
Ex 344 nm
Em 380 nm
Excimer ~ 470 nm
FRET of NBD-Rhodamine pair
NBD-Rho FRET efficiency as a function of acceptor concentration.
NBD (donor) and Rhodamine (acceptor) were labeled at the ends of HA2 FP and TMD peptides,
respectively, to examine interaction between the two molecules. Different combinations are depicted
by various curves as indicated and the dashed curve is derived from random distribution of R0 = 60 Å
donor-acceptor pair. Higher FRET efficiency from experimental data for the labeled NBD-Rho pair
than that from the theoretical computation at any given Rhodamine concentration suggests
association between TMD and FP in the membrane bilayer.
BMC Biology 2008, 6:2
FRET of Pyrene-NBD pair
FRET measurements disclose interaction between TMD and FP in an antiparallel
manner.
The efficiency of FRET between pyrene and NBD labeled to the N- and C-termini of HA2 TMD and
FP peptides in different combinations is compared to determine the orientation of the TMD:FP
complex. FRET efficiency is larger for the donor and acceptor fluorophores attached to the opposite
ends of TMD and FP. It is also noted that the interaction between FP and TMD is stronger at pH 5.0
than at 7.4 as reflected by greater transfer efficiency.
BMC Biology 2008, 6:2
FRET between NBD- and Rho-labeled lipid
The extent of membrane fusing reflected by a decrease in FRET of NBD due to
dilution of labeled phospholipids upon vesicle mixing as a function of HA2(125) concentration.
Two lipids were prepared and mixed:
labeled vesicle dispersion containing DMPC:DMPG:NBD-PE:Rho-PE
unlabelled vesicle disperion containing DMPC:DMPG.
(A) Lipid mixing as probed by the
fluorescence energy transfer between NBDand Rho-labelled lipids at pH 5.0 and 37 C
as a function of HA2(1-25) fusion peptide
concentration.
(B) The initial rate of lipid mixing taken from the
first 3 min of the mixing curves in (A). A non-linear
variation of mixing rate with peptide concentration
is an indication of cooperation of the fusion
peptide in mediating membrane mixing.
Mol. Memb. Biol. 2003, 20, 345-351
Quenching
The fluorescence quenching study monitors the accessibility
of the fluorophore to the quencher.
The data are analyzed using the Stern-Volmer equation:
F0/F = 1 + KSV·[Q]
F0 is the fluorescence intensity at the zero quencher
concentration
F is the fluorescence intensity at any given quencher
concentration [Q]
KSV represents the apparent Stern-Volmer quenching constant,
obtained from the slope of the plot of F0/F versus [Q].
Acrylamide Quenching of Trp fluorescence
Acrylamide quenching measurements indicate deep insertion of HA2 TMD into
the membrane interior. The dramatic decrease in KSV in the vesicular
dispersion compared with that in PB buffer shows that tryptophan side chains
are embedded deep into the membrane. Moreover, a twofold reduction in KSV,
as well as decreased KSV on neutralization, upon incubating in PC:PG vesicles
at pH 5.0 compared with that at pH 7.4 suggests that the TMD penetration is
deeper at acidic pH.
BMC Biology 2008, 6:2
Co2+ Quenching of NBD labeled peptide
Quenching of NBD-labeled HA2(1–25) by Co2+ in a pH cycle (5-7-5)
KSV increases with neutralization, reflecting a decrease in the
insertion depth and the KSV values are seen reversible with
respect to pH alteration
Biochem. J. 2006, 396, 557–563