Circular

1
Circular Dichroism Spectroscopy (CD)
2
Optically active sample vs. polarized light
Elliptically polarized light produced by passing
the incident light through an optically active
sample.
Incident linearly polarized light.
Resolution of linearly polarized light into
individual right-hand and left-hand circularly
polarized components.
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.
3
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 q222MMRW/10lc
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
c mg/ml l cm
Molar ellipticity q
degree cm2 dmol-1
De q /3298
Litre mol-1 cm-1 or Litre (mol residue)-1 cm-1
4
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.

5
CD Spectrum of Protein
Near-UV CD spectrum 250350 nm protein
tertiary structure Far-UV CD spectrum 190250
nm protein secondary structure ? The signal
strength in the near-UV CD region is much
weaker than that in the far-UV CD region.
6
Far-UV CD spectra
7
Near-UV CD spectrum
Biochimica et Biophysica Acta 1751, 119 139
(2005)
8
CD spectra of HIV-1 gp41 ectodomain
Electrophoresis 2008, 29, 31753182
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.
9
Fluorescence Spectroscopy
10
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).
11
Schematic of a fluorometer
A fluorometer with a 90 geometry utilizing a Xe
light source
12
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
• NBD-Rhodamine pair
• Pyrene-NBD pair
• Label on lipid
• Quenching
• Tryptophan quenching with Acrylamide
• NBD quenching with Co2

13
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.
14
Low-pH-induced conformation change of HA2
Low pH
Nature 371, 37-43 (1994)
15
Trp fluorescence of HA2 TMD peptide
Ex l 280 nm
BMC Biology 2008, 62
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.
16
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.

17
Binding of Bis-ANS to HA2(21-174)
Ex l 290 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.
18
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)-carboxytetrame
thylrhodamine 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)-carboxytetramet
hylrhodamine, succinimidyl ester, is the
amine-reactive, mixed isomer form of TAMRA.
19
R18 dequenching of HA2 fusion peptide
(A) Kinetics of HA2 FP-induced dequenching of R18
at different pH values. The pH of curves af are
indicated in panel B. (B) Wild type fusion
peptide induced percent dequenching as a function
of pH.
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.
Chem. Phys. Lipids 2000, 107, 99106
Ex l 530 nm
20
Dequenching of rhodamine labeled HA2 FP
Self-association of the 25-mer fusion peptide
analogs of HA2 in DMPCDMPG (41) 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
21
Rhodamine composition experiments
xRho-peptide/Total peptide
Rhodamine composition experiments detect tight
self-association of HA2 TMD and non-random
interaction of TMDFP 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 0.31.0 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, 62
22
NBD fluorescence
NBD 4-chloro-7-nitrobenz-2-oxa-1,3-diazole Th
e 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 lipidwater
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.
23
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, 47254736
24
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.   
25
Tb3/DPA leakage experiments
Membrane leakage experiments using Tb3/DPA assay
to monitor membrane activity of TMD, FP and
TMDFP complex of HA2. Both FP and FPTMD 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, 62
26
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 lt10
  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

27
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, 62
28
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 TMDFP 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, 62
29
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(1- 25) concentration. Two
lipids were prepared and mixed labeled vesicle
dispersion containing DMPCDMPGNBD-PERho-PE un
labelled vesicle disperion containing DMPCDMPG.
(A) Lipid mixing as probed by the fluorescence
energy transfer between NBD- and 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
30
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 KSVQ 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.
31
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 PCPG 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, 62
32
Co2 Quenching of NBD labeled peptide
Quenching of NBD-labeled HA2(125) 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, 557563