Structural changes in the Schiff base region of squid rhodopsin upon photoisomerization studied by low-temperature FTIR spectroscopy

Low-temperature Fourier transform infrared (FTIR) spectroscopy is used to study squid rhodopsin at 77 K in investigating structural changes in the Schiff base region upon photoisomerization. The analysis of O-D stretching vibrations in D(2)O revealed that there are more internal water molecules near the retinal chromophore in squid rhodopsin than in bovine rhodopsin. Among nine O-D stretching vibrations of water in squid rhodopsin, eight peaks are identical between rhodopsin and 9-cis-rhodopsin (Iso). On the other hand, the isomer-specific O-D stretch of water was observed for rhodopsin (2451 cm(-)(1)) and Iso (2382 cm(-)(1)). Low frequencies of these bands suggest that the water forms a strong hydrogen bond with a negatively charged counterion. In addition, it was suggested that the hydrogen bond of the Schiff base is weaker in squid rhodopsin than in bacteriorhodopsin and bovine rhodopsin, and squid rhodopsin possessed similar hydrogen bonding strength for the Schiff base among rhodopsin, Iso, and bathorhodopsin. Most vibrational bands in the X-D stretch region originate from water O-D or the Schiff base N-D stretches, suggesting that the hydrogen bonding network in the Schiff base region of squid rhodopsin is composed of only water molecules. On the basis of these results, we propose that squid rhodopsin possesses a "bridge" water between the Schiff base and its counterion as well as squid retinochrome [Furutani, Y., Terakita, A., Shichida, Y., and Kandori, H. (2005) Biochemistry 44, 7988-7997], which is absent in vertebrate rhodopsin [Furutani, Y., Shichida, Y., and Kandori, H. (2003) Biochemistry 42, 9619-9625].     

Localization and orientation of functional water molecules in bacteriorhodopsin as revealed by polarized Fourier transform infrared spectroscopy.

Linear dichroic difference Fourier transform infrared spectra upon formation of the M photointermediate were recorded with oriented purple membranes. The purpose was to determine the angle of the directions of the dipole moments of 1) the water molecule whose O-H stretching vibration appears at 3643 cm-1 for the unphotolyzed state and 3671 cm-1 for the M intermediate, and 2) the C=O bond of protonated Asp85 in the M intermediate. The angle of 36 degrees we find for the C=O of the protonated Asp85 in the M intermediate is not markedly different from 26 degrees for unprotonated Asp85 in the model based on cryoelectron diffraction, indicating the absence of gross orientation changes in Asp85 upon its protonation. The O-H band at 3671 cm-1 of a water molecule in the M intermediate, although its position has not determined, is fixed almost parallel to the membrane plane. For the unphotolyzed state the angle of the water O-H to the membrane normal was determined to be 60 degrees. On the basis of these data and the structural model, we place the water molecule in the unphotolyzed state at a position where it forms hydrogen bonds with the Schiff base, Asp85, Asp212, and Trp86.     

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.     

Structural changes in bacteriorhodopsin during the photocycle measured by time-resolved polarized Fourier transform infrared spectroscopy.

The structural changes in bacteriorhodopsin during the photocycle are investigated. Time resolved polarized infrared spectroscopy in combination with photoselection is used to determine the orientation and motion of certain structural units of the molecule: Asp-85, Asp-96, Asp-115, the Schiff base, and several amide I vibrations. The results are compared with recently published x-ray diffraction data with atomic resolution about conformational motions during the photocycle. The orientation of the measured vibrations are also calculated from the structure data, and based on the comparison of the values from the two techniques new information is obtained: several amide I bands in the infrared spectrum are assigned, and we can also identify the position of the proton in the protonated Asp residues.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.     

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.     

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.     

Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca2+ depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S(1)-to-S(2) transition was obtained with Ca(2+)-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca(2+) to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S(2)/S(1) FTIR spectrum upon Ca(2+) depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca(2+) depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K(+) at the Ca(2+) site. In the present study, the preparation of the Ca(2+)-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K(+). The obtained S(2)/S(1) spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm(-1) and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K(+). This result indicates that the drastic FTIR changes are a pure effect of Ca(2+) depletion, and provides solid evidence for the general view that Ca(2+) is strongly coupled with the Mn cluster.     

Structural investigation of bacteriorhodopsin and some of its photoproducts by polarized Fourier transform infrared spectroscopic methods-difference spectroscopy and photoselection

The direction of selected IR-transition moments of the retinal chromophore of bacteriorhodopsin (BR) and functional active amino acid residues are determined for light- and dark-adapted BR and for the intermediates K and L of the photocycle. Torsions around single bonds of the chromophore are found to be present in all the investigated BR states. The number of twisted single bonds and the magnitude of these torsions decreases in the order K, L, light-adapted BR, dark-adapted BR. In the last, only the C₁₄—C₁₅ single bond is twisted. The orientation of molecular planes and chemical bonds of such protein side chains, which are perturbed during the transition of light-adapted BR to the respective intermediates, are deduced and the results compared with the current three dimensional model of BR. Trp 86 and Trp 185 are found to form a rigid part of the protein, whereas Asp 96 and Asp 115 perform molecular rearrangements upon formation of the L-intermediate.     

Structural and functional implications of the instability of the ADP/ATP transporter purified from mitochondria as revealed by FTIR spectroscopy

The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies. When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates. By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process. To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein. From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands. We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

Recognition and discrimination of gases by the oxygen-sensing signal transducer protein HemAT as revealed by FTIR spectroscopy

The determination of ligand binding properties is a key step in our understanding of gas sensing and discrimination by gas sensory proteins. HemAT is a newly discovered signal transducer heme protein that recognizes O(2) and discriminates against other gases such as CO and NO. We have used FTIR spectroscopy on CO- and NO-bound sensor domain HemAT and sensor domain distal mutants Y70F, T95A, R91A, and L92A to gain insight into the structure of the iron-bound ligand at ambient temperature. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand and also allow us to investigate the communication pathway between the distal residues of the protein and the heme. We show the formation of both H-bonded and non-H-bonded conformations in the CO-bound forms. In addition, we report the presence of multiple conformations in the NO-bound forms. Such distal H-bonding is crucial for ligand binding and activation by the heme. The comparison of the O(2), NO, and CO data demonstrates that Thr95 and Tyr70 are crucial for ligand recognition and discrimination and, thus, for specific sensing of gases, and L92 is crucial for controlling the conformational changes of the Thr95 and Tyr70 residues upon NO binding.     

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.     

Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

Light-induced Fourier transform infrared difference spectroscopy has been applied to studies of ammonia effects on the oxygen-evolving complex (OEC) of photosystem II (PSII). We found that NH(3) induced characteristic spectral changes in the region of the symmetric carboxylate stretching modes (1450-1300 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra of PSII. The S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the controlled samples was very likely upshifted to 1379 cm(-1) in that of NH(3)-treated samples; however, the frequency of the corresponding S(1) carboxylate mode at 1402 cm(-1) in the same spectrum was not significantly affected. These two carboxylate modes have been assigned to a Mn-ligating carboxylate whose coordination mode changes from bridging or chelating to unidentate ligation during the S(1) to S(2) transition [Noguchi, T., Ono, T., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200; Kimura, Y., and Ono, T.-A. (2001) Biochemistry 40, 14061-14068]. Therefore, our results show that NH(3) induced significant structural changes of the OEC in the S(2) state. In addition, our results also indicated that the NH(3)-induced spectral changes of the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the temperature of the FTIR measurement. Among the temperatures we measured, the strongest effect was seen at 250 K, a lesser effect was seen at 225 K, and little or no effect was seen at 200 K. Furthermore, our results also showed that the NH(3) effects on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the concentrations of NH(4)Cl. The NH(3)-induced upshift of the 1365 cm(-1) mode is apparent at 5 mM NH(4)Cl and is completely saturated at 100 mM NH(4)Cl concentration. Finally, we found that CH(3)NH(2) has a small but clear effect on the spectral change of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. The effects of amines on the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (NH(3) > CH(3)NH(2) > AEPD and Tris) are inverse proportional to their size (Tris approximately AEPD > CH(3)NH(2) > NH(3)). Therefore, our results showed that the effects of amines on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are sterically selective for small amines. On the basis of the correlations between the conditions (dependences on the excitation temperature and NH(3) concentration and the steric requirement for the amine effects) that give rise to the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII and the conditions that give rise to the altered S(2) state multiline EPR signal, we propose that the NH(3)-induced upshift of the 1365 cm(-1) mode is caused by the binding of NH(3) to the site on the Mn cluster that gives rise to the altered S(2) state multiline EPR signal. In addition, we found no significant NH(3)-induced change in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum at 200 K. Under this condition, the OEC gives rise to the NH(3)-stabilized g = 4.1 EPR signal and a suppressed g = 2 multiline EPR signal. Our results suggest that the structural difference of the OEC between the normal g = 2 multiline form and the NH(3)-stabilized g = 4.1 form is small.     

Dihedral angles of tripeptides in solution directly determined by polarized Raman and FTIR spectroscopy

The amide I mode of the peptide linkage is highly delocalized in peptides and protein segments due to through-bond and through-space vibrationally coupling between adjacent peptide groups. J. Phys. Chem. B. 104:11316-11320) used coherent femtosecond infrared (IR) spectroscopy to determine the excitonic coupling energy and the orientational angle between the transition dipole moments of the interacting amide I modes of cationic tri-alanine in D(2)O. Recently, the same parameters were determined for all protonation states of tri-alanine by analyzing the amide I bands in the respective IR and isotropic Raman spectra (. J. Am. Chem. Soc. 119:1720-1726.). In both studies, the dihedral angles phi and psi were then obtained by utilizing the orientational dependence of the coupling energy obtained from ab initio calculations on tri-glycine in vacuo (. J. Raman Spectrosc. 29:81-86) to obtain an extended 3(1) helix-like structure for the tripeptide. In the present paper, a novel algorithm for the analysis of excitonic coupling between amide I modes is presented, which is based on the approach by Schweitzer-Stenner et al. but avoids the problematic use of results from ab initio calculations. Instead, the dihedral angles are directly determined from infrared and visible polarized Raman spectra. First, the interaction energy and the corresponding degree of wave-function mixing were obtained from the amide I profile in the isotropic Raman spectrum. Second, the depolarization ratios and the amide I profiles in the anisotropic Raman and IR-absorption spectra were used to determine the orientational angle between the peptide planes and the transition dipole moments, respectively. Finally, these two geometric parameters were utilized to determine the dihedral angles phi and psi between the interacting peptide groups. Stable extended conformations with dihedral angles in the beta-sheet region were obtained for all protonation states of tri-alanine, namely phi(+) = -126 degrees, psi(+) = 178 degrees; phi(+/-) = -110 degrees, psi(+/-) = 155 degrees; and phi(-) = -127 degrees, psi(-) = 165 degrees for the cationic, zwitterionic, and anionic state, respectively. These values reflect an extended beta-helix structure. Tri-glycine was found to be much more heterogeneous in that different extended conformers coexist in the cationic and zwitterionic state, which yield a noncoincidence between isotropic and anisotropic Raman scattering. Our study introduces vibrational spectroscopy as a suitable tool for the structure analysis of peptides in solution and tripeptides as suitable model systems for investigating the role of local interactions in determining the propensity of peptide segments for distinct secondary structure motifs.     

Increased backbone flexibility in threonine45-phosphorylated hirudin upon pH change.

Protein phosphorylation on serine/threonine side chains represents a major regulatory event in the posttranslational control of protein functionality, where it is thought to operate at the level of structural changes in the polypeptide chain. However, key questions about molecular aspects of phosphate ester induced conformational alterations remain open. Among these concerns are the radius of action of the phosphate ester group, its effective ionic state, and its interplay with distinct bonds of the polypeptide chain. Primarily to define short-range effects upon threonine phosphorylation, the native 65 amino acid protein hirudin, conformationally restrained by a proline flanking the pThr(45) site and three intramolecular disulfide bonds, was structurally characterized in both the phosphorylated and the unphosphorylated state in solution. Circular dichroism and hydrogen exchange experiments (MALDI-TOF) showed that structural changes were caused by Thr(45)-Pro(46) phosphorylation only when the phosphate ester group was in its dianionic state. The spatial arrangement of the amino acids, monitored by 1H NMR spectroscopy, appears to be affected within a radius of about 10 A around the pThr(45)-OgammaH, with phosphorylation resulting in a loss of structure and increased flexibility within a segment of at least seven amino acid residues. Thus, the transition from the monoanionic to the dianionic phosphate group over the pH range 5.2-8.5 represents a general phosphorylation-dependent conformational switch operating at physiological pH values.