Understanding Circular Dichroism and Fluorescence Spectroscopy

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 q x 100 x Mr c x l 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 c: mg/ml l: cm Molar ellipticity: [q] = degree cm2 dmol-1 De = [q] /3298 Litre mol-1 cm-1 orLitre (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 -- thermalstability, 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: 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.

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 (B) Near-UV spectra (A) Far-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.

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 38 55 146 | 153 76 129 113 105 Low pH Nature 371, 37-43 (1994)

15. 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.

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 highlyself-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.

19. 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. where Ftand 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. Lipids2000, 107, 99–106

20. 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. Acta2003, 1612, 41– 51

21. 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

22. 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.

23. NBD fluorescent spectra of ASLV-IFP Lipid binding of NBD-IFP (internal fusion peptide) of the avian sarcoma and leucosis virus (ASLV) envelope glycoproteinat 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

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 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

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 <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

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, 6:2

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 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

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 DMPC:DMPG:NBD-PE:Rho-PE unlabelled vesicle disperion containing DMPC:DMPG. (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 fluorophoreto 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].

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 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

32. 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 theKSV values are seen reversible with respect to pHalteration Biochem. J. 2006, 396, 557–563