Intensity of cooperative vibronic transitions in the Eu(III), Gd(III) and Tb(III) formates spectra

The luminescence and absorption spectra of the lanthanide ions in solids and coordination compounds are characterized by sharp pure electronic lines, which are accompanied by much weaker lines of vibronic transitions. The vibronic spectroscopy is a good probing tool for investigations of the properties of surrounding ion ligands. The lanthanides formates are efficient luminescent crystals and can be viewed as the elementary type in the whole class of the oxygen-containing lanthanide coordination compounds. The intensity of vibronic transitions in spectra of luminescence and excitation europium (⁵D₀→⁷F₂, ⁷F₀→⁵D₂), terbium (⁷F₆→⁵D₄), gadolinium (⁶P_7/2→⁸S_7/2) in anhydrous formates of the type Ln(HCOO)₃ (Ln = Eu, Tb, Gd) and Y(HCOO)₃.2H₂O doped with Eu³⁺ and Tb³⁺ (C ~1 mol%) are reported. Also, the infrared and Raman spectra were obtained for the same compounds. Related integral intensity vibronic sidebands depend on the type of electronic transition of the same ion and varies for the same electronic transitions in different crystals. The obtained experimental data referring to the rate constants of vibronic transitions and intensity distribution in vibronic spectra on normal vibrations of the formate groups are in agreement with the predictions based on the Stavola–Dexter theory of cooperative vibronic transitions.     

A study of protein–carotenoid interactions in the astaxanthin-protein crustacyanin by absorption and Stark spectroscopy; evidence for the presence of three spectrally distinct species

Molecular mechanisms underlying the peculiar spectral properties of the carotenoid astaxanthin in α-crustacyanin, the blue carotenoprotein isolated from the exoskeleton of the lobster Homarus gammarus, were investigated by comparing the basic electrooptical parameters of astaxanthin free in vitro with those of astaxanthin in the complex. Absorption and electroabsorption (Stark effect) spectra were obtained for α-crustacyanin in low-temperature glasses to provide information about the molecular interactions that lead to the large bathochromic shift of the spectra resulting from this complexation. The low-temperature spectra reveal the presence of at least three spectral forms of α-crustacyanin, with vibronic (0–0) transitions at 14 000 cm⁻¹, 13 500 cm⁻¹ and 11 600 cm⁻¹ (corresponding to approximately 630, 660 and 780 nm, respectively, at room temperature) and with relative aboundance 85%, 10% and 5%. The longer wavelength absorbing species have not previously been detected. The changes in polarizability and in permanent dipole moments associated with the S₀→S₂ electronic transition for all these forms are about 1.5 times larger than for isolated astaxanthin. The results are discussed with reference to the symmetric polarization model for astaxanthin in α-crustacyanin.     

Multinuclear NMR of cis-dicarbonylbis(dithiocarbamato)iron(II) complexes in chloroform solution

The ¹³C and ¹⁵SN NMR spectra of eleven cis-Fe(S₂CNRR′)₂(CO)₂ complexes, where R and R′ are organic substituents, have been measured at ambient temperature in CDCl₃ (0.08–0.16 M). The ¹³C absorptions for the carbonyl ligands correlate well with the force constants for the CO stretching vibrations in CHCl₃ solution. Each of the parameters (¹³CO absorption and k_cis for CO) correlate well with the aqueous solution pK_a for⁺H₂NRR′, corrected for the phenyl-containing substituents, high pK_a values corresponding to high ¹³CO absorptions and low k_cis CO force constants. [p ]Evidence was found in the ¹³C NMR spectra for hindered rotation about the CN bond in S₂CNC₂ in complexes with higher pK_a(corr) values and in the ¹³C spectra of the corresponding thiuram disulfides. [p ]The ¹⁵N (natural abundance) NMR spectra for each of the complexes was determined. Each revealed a single sharp absorption in a region of the ¹⁵N NMR spectrum which indicates substantial CN double bond character, as one would expect for coordinated dithiocarbamate ligands.     

Light- and MgCl2-dependent characteristics of four chlorophyll-protein complexes isolated from the marine dinoflagellate,Glenodinium sp.

Chlorophyll-protein complexes previously isolated from low-light (80 μE·m⁻²·s⁻¹) log cultures of the marine dinoflagellate, Glenodinium sp., were further characterized. SDS solubilization in combination with polyacrylamide gel electrophoresis in the presence of Deriphat 160-C resolved four discrete chlorophyll-protein bands. In order to elucidate the functional role of Glenodinium sp., room-temperature absorption and fluorescence spectra, protein composition, and pigment molar ratios were obtained for each complex. Results indicated that complex I was analogous to the green plant Photosystem I complex and was also associated with light-harvesting chlorophyll c₂. Complex II was highly enriched in chlorophyll c₂, devoid of peridinin, and demonstrated energy transfer from chlorophyll c to chlorophyll a within the complex, indicating the presence of a light-harvesting component. Based on peridinin: chlorophyll a ratios and fluorescence excitation spectra analyses for complexes III and IV, it was concluded that these complexes contained functional peridinin-chlorophyll a-protein complexes. Changing the ionic environment during isolation of the complexes, or altering the growth irradiance of Glenodinium sp. cultures, resulted in a significant alteration of distribution of chlorophyll a among the chlorophyll-protein complexes.     

Absorption and fluorescence spectra of chlorophyll-proteins isolated from Euglena gracilis

A spectroscopic study of chlorophyll-protein complexes isolated from Euglena gracilis membranes was carried out to gain information about the state of chlorophyll in vivo and energy transfer in photosynthesis. The membranes were dissociated by Triton X-100 and separated into fractions by sucrose gradient centrifugation and hydroxyapatite chromatography. Four different types of chlorophyll-protein complexes were distinguished from each other and from detergent-solubilized chlorophyll in these fractions by examination of their absorption, fluorescence excitation (400–500 nm) and emission spectra at low temperature. These types were: (1). A mixture of antenna chlorophyll a- and chlorophyll ab-proteins with an absorption maximum at 669 and emission at 682 nm; (2) a P-700-chlorophyll a-protein (chlorophyll: P-700 = 30 : 1), termed CPI with an absorption maximum at 676 nm and emission maxima at 698 and 718 nm; (3) a second chlorophyll a-protein (CPI-2) less enriched in P-700, with an absorption maximum at 676 nm and emission maxima at 680, 722 and 731 nm; (4) a third chlorophyll a-protein (CPa₁) with no P-700, absorption maxima at 670 and 683 nm, and an unusually sharp emission maximum at 687 nm. Treatment of CPa₁ with sodium dodecyl sulfate drastically altered its spectroscopic properties indicating that at least some chlorophyll-proteins isolated with this detergent are partially denatured. The results suggest that the complex absorption spectra of chlorophyll in vivo are caused by varying proportions of different chlorophyll-protein complexes, each with different groups of chlorophyll molecules bound to it and making up a unique entity in terms of electronic transitions.     

Purification and identification of chlorophyll c1 from the green alga Mantoniella squamata

The prasinophycean alga Mantoniella squamata contains besides chlorophyll a and b a third chlorophyll c-like pigment in its light-harvesting antenna. This third chlorophyll was purified by reverse phase and polyethylene chromatography in order to identify its chemical structure. The absorption and fluorescence spectra were measured not only from the doubly purified pigment, but also from its Mg-free derivates. The spectra were compared with those of authentic chlorophyll c and of Mg-2,4-desethyl-2,4-divinylpheoporphyrin a₅ monomethyl ester which was isolated from Rhodobacter capsulata. The results show that the pigment from Mantoniella agrees best with chlorophyll c₁. In order to clarify the spectral data, chlorophyll c₁ and c₂, the pigment from Mantoniella and Mg-2,4-desethyl-2,4-divinylpheoporphyrin a₅ monomethyl ester were resolved by polyethylene chromatography. The chromatographic analysis clearly shows that the pigment from Mantoniella comigrates with chlorophyll c₁ and not with the bacterial pigment or chlorophyll c₂. Mantoniella is the first organism which has been demonstrated to contain chlorophyll a, b and c.     

Photochemical properties of mesophyll and bundle sheath chloroplasts of maize

Several photochemical and spectral properties of maize (Zea mays) bundle sheath and mesophyll chloroplasts are reported that provide a better understanding of the photosynthetic apparatus of C₄ plants. The difference absorption spectrum at 298 K and the fluorescence excitation and emission spectra of chlorophyll at 298 K and 77 K provide new information on the different forms of chlorophyll a in bundle sheath and mesophyll chloroplasts: the former contain, relative to short wavelength chlorophyll a forms, more long wavelength chlorophyll a form (e.g. chlorophyll a 693 and chlorophyll a 705) and less chlorophyll b than the latter. The degree of polarization of chlorophyll a fluorescence is 6% in bundle sheath and 4% in mesophyll chloroplasts. This result is consistent with the presence of relatively high amounts of oriented long wavelength forms of chlorophyll a in bundle sheath compared to mesophyll chloroplasts. The relative yield of variable, with respect to constant, chorophyll a fluorescence in mesophyll chloroplasts is more than twice that in bundle sheath chloroplast. Furthermore, the relative yield of total chlorophyll a fluorescence is 40% lower in bundle sheath compared to that in mesophyll chloroplasts. This is in agreement with the presence of the higher ratio of the weakly fluorescent pigment system I to pigment system II in bundle sheath than in mesophyll chloroplast. The efficiency of energy transfer from chlorophyll b and carotenoids to chlorophyll a are calculated to be 100 and 50%, respectively, in both types of chloroplasts. Fluorescence quenching of atebrin, reflecting high energy state of chloroplasts, is 10 times higher in mesophyll chloroplasts than in bundle sheath chloroplasts during noncyclic electron flow but is equal during cyclic flow. The entire electron transport chain is shown to be present in both types of chloroplasts, as inferred from the antagonistic effect of red (650 nm) and far red (710 nm) lights on the absorbance changes at 559 nm and 553 nm, and the photoreduction of methyl viologen from H₂O. (The rate of methyl viologen photoreduction in bundle sheath chloroplasts was 40% of that of mesophyll chloroplasts.)     

Re-examination of the emission properties of alkoxy- and thioalkyl-substituted phthalocyanines

The role of n → π^∗ transitions in the optical spectra of alkoxy- and thioalkyl-substituted phthalocyanines with peripheral substituents is re-examined, based on a detailed analysis of UV-visible absorption, magnetic circular dichroism (MCD) and fluorescence emission spectral data and the results of density functional theory (DFT) and time dependent-density functional theory (TD-DFT) calculations. The nπ^∗ excited states associated with the lone pairs of the peripheral heteroatoms have been proposed as the origin of a second emission peak observed in the 400-600 nm region, which has been assigned as S₂ emission, since S₁ fluorescence associated with the Q band is observed in the near IR region. Our results demonstrate that emission from a photodecomposition product can fully account for this violet emission and that reports of S₂ emission for peripherally substituted phthalocyanines and porphyrazines should be treated with caution.     

Fluorescence characteristics of photoautotrophic soybean cells

We report here the first measurements on chlorophyll (Chl) a fluorescence characteristics of photoautotrophic soybean cells (cell lines SB-P and SBI-P). The cell fluorescence is free from severe distortion problems encountered in higher plant leaves. Chl a fluorescence spectra at 77 K show, after correction for the spectral sensitivity of the photomultiplier and the emission monochromator, peaks at 688, 696 and 745 nm, representing antenna systems of photosystem II-CP43 and CP47, and photosystem I, respectively. Calculations, based on the complementary area over the Chl a fluorescence induction curve, indicated a ratio of 6 of the mobile plastoquinone (including Q_B) to the primary stable electron acceptor, the bound plastoquinone Q_A. A ratio of one between the secondary stable electron acceptor, bound plastoquinone Q_B, and its reduced form Q_B ^- was obtained by using a double flash technique. Owing to this ratio, the flash number dependence of the Chl a fluorescence showed a distinct period of four, implying a close relationship to the S state of the oxygen evolution mechanism. Analysis of the Q_A ^- reoxidation kinetics showed (1) the halftime of each of the major decay components ( 300 s fast and 30 ms slow) increases with the increase of diuron and atrazine concentrations; and (2) the amplitudes of the fast and the slow components change in a complementary fashion, the fast component disappearing at high concentrations of the inhibitors. This implies that the inhibitors used are able to totally displace Q_B. In intact soybean cells, the relative amplitude of the 30 ms to 300 s component is higher (40:60) than that in spinach chloroplasts (30:70), implying a larger contribution of the centers with unbound Q_B. SB-P and SBI-P soybean cells display a slightly different sensitivity of Q_A ^- decay to inhibitors.Abbreviations CA complementary area over fluorescence induction curve - Chl chlorophyll, diuron - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F _m maximum chlorophyll a fluorescence - F ₀ minimum chlorophyll a fluorescence - F _v = F _t-F₀ - where F _v = variable chlorophyll a fluorescence - and F_t = chlorophyll a fluorescence at time t - PS II photosystem II - Q _a primary (plastoquinone) electron acceptor of PS II - Q _b secondary (plastoquinone) electron acceptor of PS II - t₅₀ the time at which the concentration of reduced Q _a is 50% of that at its maximum value     

Phytol as one of the determinants of chlorophyll interactions in solution

Optical absorption and fluorescence parameters of chlorophyll a and the phytol-free chlorophyllide a, as well as of their Mg-depleted derivatives, were compared in a series of organic solvents. In contrast to prevailing opinion, the spectral properties of chlorophyll are not indifferent to the removal of phytol. The electronic absorption spectra of chlorophyll a and chlorophyllide a differ and display a different dependence on the nature of the solvent, which cannot be explained solely by the location of a charged carboxylic group in the proximity of the π– electron system. In fact, measurements in media of varying basicity show that deprotonation of the free carboxylic group in chlorophyllide, i.e., the presence of a negative point charge near the macrocycle, has no effect on pigment absorption spectra. Analysis of the solvent effect on the Q_Y energies in terms of solvent polarity reveals that the phytyl moiety perturbs the spectral features of chlorophyll, mainly due to its interactions with the pigment solvation shell. The phytyl residue might also be thus partly involved in controlling the central metal ligation in chlorophylls. This influence of phytol on the spectral features of chlorophyll should be taken into account when comparing the spectra in solution with various spectral forms of chlorophyll in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

How the molecular structure determines the flow of excitation energy in plant light-harvesting complex II

Excitation energy transfer in the light-harvesting complex II of higher plants is modeled using excitonic couplings and local transition energies determined from structure-based calculations recently (Müh et al., 2010). A theory is introduced that implicitly takes into account protein induced dynamic localization effects of the exciton wavefunction between weakly coupled optical and vibronic transitions of different pigments. Linear and non-linear optical spectra are calculated and compared with experimental data reaching qualitative agreement. High-frequency intramolecular vibrational degrees of freedom are found important for ultrafast subpicosecond excitation energy transfer between chlorophyll (Chl) b and Chla, since they allow for fast dissipation of the excess energy. The slower ps component of this transfer is due to the monomeric excited state of Chlb 605. The majority of exciton relaxation in the Chla spectral region is characterized by slow ps exciton equilibration between the Chla domains within one layer and between the lumenal and stromal layers in the 10-20 ps time range. Subpicosecond exciton relaxation in the Chla region is only found within the terminal emitter domain (Chls a 610/611/612) and within the Chla 613/614 dimer. Deviations between measured and calculated exciton state life times are obtained for the intermediate spectral region between the main absorbance bands of Chla and Chlb that indicate that besides Chlb 608 another pigment should absorb there. Possible candidates, so far not identified by structure-based calculations, but by fitting of optical spectra and mutagenesis studies, are discussed. Additional mutagenesis studies are suggested to resolve this issue.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Photoadaptation in marine phytoplankton : changes in spectral absorption and excitation of chlorophyll a fluorescence

The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations.     

Spectral and photochemical properties of borohydride-treated D1-D2-cytochrome b-559 complex of photosystem II

The D1-D2-cytochrome b-559 reaction center complex of photosystem II with an altered pigment composition was prepared from the original complex by treatment with sodium borohydride (BH⁻₄). The absorption spectra of the modified and original complexes were compared to each other and to the spectra of purified chlorophyll a and pheophytin a (Pheo a) treated with BH⁻₄ in methanolic solution. The results of these comparisons are consistent with the presence in the modified complex of an irreversibly reduced Pheo a molecule, most likely 13¹-deoxo-13¹-hydroxy-Pheo a, replacing one of the two native Pheo a molecules present in the original complex. Similar to the original preparation, the modified complex was capable of a steady-state photoaccumulation of Pheo⁻ and P680⁺. It is concluded that the pheophytin a molecule which undergoes borohydride reduction is not involved in the primary charge separation and seems to represent a previously postulated photochemically inactive Pheo a molecule. The Q_y and Q_x transitions of this molecule were determined to be located at 5°C at 679.5–680 nm and 542 nm, respectively.     

Part of the fluorescence of chlorophyll a may originate in excited triplet states

Absorption and fluorescence spectra of chlorophyll a have been analyzed on the basis of an extended version of Kennard–Stepanov (KS) theory. It is proposed that at least one new electronic state lies just below the normal S₁ – S₀ transition (Q_y), borrowing approximately 2–4% of its strength and contributing to the fluorescence in the tail. The KS anomalies leading to this hypothesis occur in a wide variety of cases, including chlorophyll a in solution and protein-bound chlorophyll a, suggesting that the phenomenon is an intrinsic property of the molecule. Natural candidates for the new state(s) are the second and third triplet states. The relationship of the fluorescence excitation spectrum to KS theory is investigated and applied to explain a red drop in yield.     

In vitro preparation and characterization of a 700 nm absorbing chlorophyll-water adduct according to the proposed primary molecular unit in photosynthesis

1. This study characterizes chlorophyll a-H₂O adducts in vitro in order to establish their generic relationship to the recently proposed [15, 18–20, 31] primary molecular adduct in photosynthesis. The effects of water titration and temperature on the absorption, fluorescence, excitation, and redox properties of the various in vitro chlorophyll a aggregate species are investigated.2. From fluorescence measurements, we conclude that the driest chlorophyll a sample contains an equimolar amount of water. This conclusion is consistent with earlier experimental work [2, 3, 14, 17, 31], and clarifies the origin of the controversial [15] Katz model [14] of chlorophyll a-H₂O interactions.3. With increasing water concentration or as the temperature is lowered below room temperature, the A-663 monohydrate chlorophyll a · H₂O (species absorbing at 663 nm) is favored at the expense of the A-678 anhydrous aggregate according to the equilibrium 2H₂O + chlorophyll a₂ai 2 chlorophyll a · H₂O. Under excess water conditions, A-663 is converted to A-743 (chlorophyll a · 2H₂O)_n.4. On slow sample cooling to T ? 200 °K, we observe the growth of A-700 at the expense of A-663. There is a direct correspondence between the increasing (decreasing) absorption by A-700 (A-663) and increasing (decreasing) fluorescence at 720 nm (664 nm).5. It is concluded that A-700 is most probably the dimer participating in the equilibrium 2 chlorophyll a · H₂O ai (chlorophyll a · H₂O)₂. The A-700 band consists of two exciton components (separated by ≈ 280 cm^?1) that are interpretable in terms of the dimeric origin of A-700.6. The deconvoluted A-700 absorption spectrum and the excitation spectrum of the 720 nm fluorescence are compared with the light-minus-dark spectra of P-700.7. It is found that A-700 is reversibly bleached by I₂ (E₀ = 0.54 V). The significance of this observation is discussed in terms of the redox properties of monomeric chlorophyll a and P-700.     

The photochemical and fluorescence properties of whole cells,spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured: Photosynthesis (O₂ evolution) in whole cells; Hill reaction (O₂ evolution) with Fe(CN)₆^3? and NADP as electron acceptors (Photosystem II and Photosystem II+Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern.On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90 % (10 %) of the chlorophyll a, 90 % (10 %) of the carotenoids and 15 % (85 %) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments: they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction.The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20–40 %) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion.The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component.The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.     

Conformational characteristics of polypeptides containing isolated L-proline residues with cis peptide bonds

The conformational characteristics of the peptide sequence X-l-Pro, where X  Gly or l-Ala and the peptide bond joining X and l-Pro is cis, are evaluated. Semi-empirical potential functions are used to estimate the contributions to the conformational energy made by the non-bonded van der Waals' and electrostatic interactions and the intrinsic torsional potentials about the NC^a and C^aC′ bonds. Rotations φ₁ and ψ₁ about the NC^a and C^aC′ bonds in residue X and rotation ψ₂ about the C^aC′ bond in l-Pro are permitted, while the angle of rotation φ₂ about the NC^a bond in l-Pro is fixed at 120 ° by the pyrrolidine ring. The presence of the cis peptide bond connecting X and l-Pro renders the backbone rotations φ₁, ψ₁ in X dependent upon the rotation ψ₂ about the C^aC′ bond in l-Pro. (Interdependence of rotations in neighboring residues joined by a cis peptide bond was previously observed in l-alanine oligomers.) The number of energetically allowed conformations for the Gly and l-Ala residues preceding a cis peptide bond l-Pro residue are found to be substantially reduced from those permitted when the peptide bond is trans or when l-Pro is replaced by an amino acid residue. On the other hand, ψ₂ = 100 to 160 ° (cis′) and 300 to 0 ° (trans′) are found to be the lowest energy conformations of the l-Pro residue irrespective of the cis or trans conformation of the X-l-Pro peptide bond.     

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

The absorption spectra of N-acetyl-l-tryptophanamide in various solvents were resolved into the sums of the ¹L_a and ¹L_b components. The relative intensities of the 0-0 transitions of the ¹L_b bands correlate linearly with the solvent polarity values (). A novel strategy that uses a set of the experimental ¹L_b bands was employed to resolve the near-UV circular dichroism (CD) spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence and structural data of TL. Analysis of the ¹L_b bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The dimethyl sulfoxide (DMSO)-like ¹L_b band of Trp CD spectra may be used as a “fingerprint” to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method, which we call site-directed circular dichroism, is broadly applicable to various proteins to obtain the position-specific data.