In situ measurement of conformational changes in proteins at liquid interfaces by circular dichroism spectroscopy

A new circular dichroism (CD) spectroscopy technique for studying conformational changes in proteins in situ at the air-water interface is described. By using this technique, conformations of four proteins, viz., beta-casein, bovine serum albumin (BSA), lysozyme, and fibrinogen in the adsorbed state at the air-water interface have been studied. beta-Casein, which is predominantly in a disordered state in solution, assumes a beta-sheet conformation at the air-water interface. On the other hand, lysozyme and fibrinogen, which are alpha+beta-type proteins in solution, become beta-type proteins by completely transforming their alpha-helix structure into beta-sheets. Bovine serum albumin, which is an alpha-type protein in solution, loses its alpha-helix and becomes a disordered protein at the air-water interface. The results indicated that during unfolding and film formation at the interface, structural changes in proteins, regardless of their initial native state, follow the course of increasing beta-sheet and disordered structure and decreasing alpha-helix content. Although this seems to be the general trend, the exceptional case of BSA suggests, however, that this is not universal.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

Characterization of the emulsification properties of 2S albumins from sunflower seed

The ability of 2S albumins from sunflower seeds to stabilize oil-in-water emulsions has been investigated, demonstrating that one of the proteins (SFA8) effectively stabilizes emulsions, while another (SF-LTP) does not stabilize emulsions. The surface tension and surface dilation viscosity of these two proteins were measured, rationalizing the emulsifying ability of SFA8 in terms of its ability to form a strongly elastic monolayer at interfaces. The secondary structure changes that occur upon adsorption of SFA8 to the oil/water interface have also been studied by fluorescence, circular dichroism (CD), and Fourier-transform infrared (FT-IR) spectroscopy. It was found that the beta-sheet content of the protein increased upon adsorption at the expense of alpha-helix and random structure. Moreover, FT-IR measurements indicate the presence of intermolecular beta-sheet formation upon adsorption. Fluorescence studies with an oil-soluble fluorescence quencher indicate that the single tryptophan residue present in SFA8 may become located in the oil-phase of the emulsion. This residue is thought to be partially buried in the native protein, and these data suggest that changes in the polypeptide region flanking this residue may play an important role in the molecular rearrangement that occur on or following adsorption to the oil/water interface.     

DEPOLARIZATION OF MOUSE MYELOMA CELL MEMBRANES DURING PHOTODYNAMIC ACTION

The effect of photodynamic action on plasma membranes was examined using a fluorescent potentiometric indicator [di-SBA-C2(3)] to measure alterations in the plasma membrane potential of mouse myeloma cells treated with zinc phthalocyanine sulfonate and light. Plasma membrane depolarization was observed to be an early event in photodynamic action, showing both photosensitizer concentration and light dose dependence. Depolarization occurred while membrane integrity was retained and appears to be an early event preceding cell death.     

Selective Examination of Plasma Membrane–Associated Photosensitizers Using Total Internal Reflection Fluorescence Spectroscopy: Correlation between Photobleaching and Photodynamic Efficacy of Protoporphyrin IX

Fluorescence spectra, fluorescence decay kinetics, photobleaching kinetics and photodynamic efficacy of protoporphyrin IX (PP) were investigated in endothelial cells in vitro after different incubation times. Fluorescence spectra and photobleaching kinetics were determined during total internal reflection (TIR) illumination or epiillumination. Because penetration depth of the excitation light during TIR illumination was limited to about 100 nm, plasma membrane-associated PP was almost selectively examined. Spectra obtained by TIR fluorescence spectroscopy (FS) showed a very low background, where-as spectra obtained by epi-illumination exhibited considerable background by autofluorescence and scattered light. For photobleaching kinetics during TIR illumination after 1 h or 24 h incubation, a biexponential fluorescence decrease was observed with a rapidly and a slowly bleaching portion. After 1 h incubation, the rapidly bleaching portion was the predominant fraction, whereas after 24 h incubation comparable relative amounts of the rapidly and slowly bleaching portion were determined. The rapidly and slowly bleaching portion were assigned to PP monomers and aggregated species in close vicinity to the plasma membrane. Fluorescence decay measurements after epi-illumination support the decrease of PP monomers within the whole cell with increasing incubation time. In contrast to TIR illumination, photobleaching of PP during epi-illumination was characterized by slow monoexponential fluorescence decrease after 1 h or 24 h incubation. Photodynamic efficacy of PP using epi-illumination was found to depend strongly on incubation time. Considerable cell inactivation was determined for short incubation times (1 h or 3 h), whereas photodynamic efficacy was diminished for longer incubation times. Reduced photodynamic efficacy after long incubation times was assigned to the lower amount of photodynamically active monomers determined close to the plasma membrane as well as within the whole cell. In conclusion, TIRFS measurements are suggested to be an appropriate tool for the examination of the plasma membrane-associated photosensitizer fraction in living cells.     

Photo-responsive NIR-II biomimetic nanomedicine for efficient cancer-targeted theranostics

In pancreatic cancer, the special barrier system formed by a large number of stromal cells severely hinders drug penetration in deep tumor tissues, resulting in low treatment efficiency. Cell membrane protein-camouflaged liposomal nanomedicines have cancer cell targeting abilities, whereas near-infrared two-zone (NIR-II) fluorescence imaging can achieve deep tissue penetration due to its long light wavelength (1,000–1,700 nm). To combine the cell membrane-based biomimetic technology with NIR-II fluorescence imaging, we constructed a biomimetic nanomedicine (BLIPO-I/D) by camouflaging indocyanine green-doxorubicin (ICG-DOX) liposomes with SW1990 pancreatic cancer cell membrane. The nanomedicine exhibited light-controlled DOX release and high pancreatic cancer treatment efficiency in vitro and in vivo. BLIPO-I/D showed the ability of targeted delivery of a large number of liposomes to pancreatic tumor tissues through homologous targeting of SW1990 cell membranes, which increased the NIR-II fluorescence imaging intensity. Irradiation of the liposomes taken up by pancreatic tumor tissues with near-infrared light (808 nm) triggered the rapid release of DOX from the liposomes, induced the photothermal and photodynamic effects of ICG, which exerted anti-tumor effects. Therefore, the fabricated biomimetic liposomal nanomedicine BLIPO-I/D is expected to achieve precise theranostics of pancreatic cancer.     

Printing biomacromolecules on a bovine serum albumin precursor layer

Various biomacromolecules including proteins and polysaccharides are printed on a substrate capped with a bovine serum albumin (BSA) precursor layer to create clear co-patterns of these molecules. Characterizations by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) demonstrate the successful production and clear boundaries of the co-patterns. Rinsing the BSA-adsorbed substrate and the biomacromolecules-inked stamp before microcontact printing (microCP) is crucial for the creation of clear and stable co-patterns. The patterns are mainly stabilized by electrostatic interactions and van der Waals forces. Characterizations by ellipsometry, UV-Vis and fluorescence spectroscopy reveal that printing by a flat PDMS stamp yields a denser layered structure of proteins with a higher amount than that of adsorbed proteins. By printing, however, a lower enzymatic catalytic activity for horseradish peroxidase (HRP) or binding capability for avidin (both normalized to amount) is determined. A conformational transition from alpha-helix to beta-sheet of HRP is observed by ATR-IR. By contrast, a BSA precursor layer can effectively improve the functionality of the printed HRP or avidin and preserve the original conformation of the proteins, although the absolute transferred amount of these proteins is decreased.     

Tracking fiber formation in human islet amyloid polypeptide with automated 2D-IR spectroscopy

Amyloid forming proteins have been implicated in many human diseases. The kinetics of amyloid fiber formation are of particular interest because evidence points to intermediate folding structures as potential cytotoxic species. The standard methods for monitoring the kinetics are to use fluorescence or circular dichroism spectroscopy, which do not uniquely resolve secondary structures. In this work, we use a new technology for rapidly scanning 2D-IR spectra that allows us to follow the fiber formation kinetics of the human islet amyloid polypeptide (hIAPP) that is involved in type II diabetes. Spectroscopic markers are identified that uniquely monitor random coil versus beta-sheet secondary structures as well as probe beta-sheet elongation and stacking. Our measurements provide more rigorous kinetics for the secondary structure evolution of amyloid formation than is available with other techniques.     

Physicochemical mechanism for the enhanced ability of lipid membrane penetration of polyarginine

Arginine-rich, cell-penetrating peptides (e.g., Tat-peptide, penetratin, and polyarginine) are used to carry therapeutic molecules such as oligonucleotides, DNA, peptides, and proteins across cell membranes. Two types of processes are being considered to cross the cell membranes: one is an endocytic pathway, and another is an energy-independent, nonendocytic pathway. However, the latter is still not known in detail. Here, we studied the effects of the chain length of polyarginine on its interaction with an anionic phospholipid large unilamellar vesicle (LUV) or a giant vesicle using poly-l-arginine composed of 69 (PLA69), 293 (PLA293), or 554 (PLA554) arginine residues, together with octaarginine (R8). ζ-potential measurements confirmed that polyarginine binds to LUV via electrostatic interactions. Circular dichroism analysis demonstrated that the transition from the random coil to the α-helix structure upon binding to LUV occurred for PLA293 and PLA554, whereas no structural change was observed for PLA69 and R8. Fluorescence studies using membrane probes revealed that the binding of polyarginine to LUV affects the hydration and packing of the membrane interface region, in which the degree of membrane insertion is greater for the longer polyarginine. Isothermal titration calorimetry measurements demonstrated that although the binding affinity (i.e., the Gibbs free energy of binding) per arginine residue is similar among all polyarginines the contribution of enthalpy to the energetics of binding of polyarginine increases with increasing polymer chain length. In addition, confocal laser scanning microscopy showed that all polyarginines penetrate across giant vesicle membranes, and the order of the amount of membrane penetration is R8 ≈ PLA69 < PLA293 ≈ PLA554. These results suggest that the formation of α-helical structure upon lipid binding drives the insertion of polyarginine into the membrane interior, which appears to enhance the membrane penetration of polyarginine.     

Interaction of chlorin p6 with bovine serum albumin and photodynamic oxidation of protein

The binding of chlorin p6, a photosensitizer having basic tetrapyrrole structure, to bovine serum albumin (BSA) and oxidation of the protein following photodynamic treatment is studied. The Stern-Volmer plot indicates that binding of chlorin p6 to BSA was of single class. Binding parameters, binding association constant and number of binding sites, were found to be 1.62+/-0.27 x 10(5)M(-1) and 1.086+/-.019, respectively. Photodynamic oxidation of protein was studied by (i) loss of intrinsic fluorescence of protein, (ii) protein carbonyl formation, (iii) protein hydroperoxide (iv) formation of TCA soluble amino groups and (v) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Intrinsic protein fluorescence was observed to decrease almost linearly as a function of irradiation time at a fixed concentration of chlorin p6 and with increasing concentration of chlorin p6 at fixed time of irradiation. Protein carbonyl and hydroperoxide formation was found to increase with increasing photodynamic treatment. No significant increase in 5% TCA soluble amino groups was observed. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reveals that photodynamic treatment of BSA in presence of chlorin p6, rose bengal and riboflavin causes non-specific fragmentation of protein. Photodynamic carbonyl formation by chlorin p6 was not inhibited by sodium formate (100 mM) or mannitol (25 mM) but was significantly inhibited by sodium azide (2 mM). Protein carbonyl formation increased almost 90% when H2O was replaced by D2O. The results show that chlorin p6 induced photodynamic oxidation of BSA was mainly mediated by singlet oxygen.     

A comparative study of the cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl) chlorin encapsulated in surface-modified submicronic oil/water carriers in HT29 tumor cells

The poor selectivity of photosensitizers for tumor tissue remains a drawback in photodynamic therapy (PDT) and could be improved by adapted formulations. The cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl)chlorin (mTHPC) encapsulated in submicronic colloidal carriers have been studied in macrophage-like J774 cells and HT 29 human adenocarcinoma cells. Nanocapsules with an external layer made of poly(D,L lactic acid) (PLA NCs), PLA grafted with polyethylene glycol (PLA-PEG NCs), PLA coated with poloxamer 188 (polox PLA NCs) and oil/water nanoemulsion (NE) have been examined. The cellular uptake by J774, as determined by microspectroflorimetry, is reduced with mTHPC encapsulated into surface-modified NCs--PLA-PEG and polox PLA--compared with naked PLA, indicating a possible limitation of the clearance of such carriers by the reticuloendothelial system. Encapsulation also modifies the interaction between mTHPC and HT29 cells. Compared with the manufacturer's solution (PEG, ethanol, water), the cellular uptake is strongly reduced. However, the HT29 phototoxicity is much less affected and a protecting effect against plasma proteins is observed. Fluorescence microscopy reveals a specific punctate fluorescence pattern with PLA-PEG and polox PLA NCs in contrast to a more diffuse distribution with NE and solution, indicating that photodamage targeting could be different. These findings suggest that photosensitizers encapsulated into surface-modified nanocapsules could be a promising approach for improving PDT efficacy and this has to be confirmed in vivo.     

Long-Circulating Prostate-Specific Membrane Antigen-Targeted NIR Phototheranostic Agent

Targeted photodynamic therapy (PDT) combined with image-guided surgical resection is a promising strategy for precision cancer treatment. Prostate-specific membrane antigen (PSMA) is an attractive target due to its pronounced overexpression in a variety of tumors, most notably in prostate cancer. Recently, we reported a pyropheophorbide-based PSMA-targeted agent, which exhibited long plasma circulation time and effective tumor accumulation. To further advance PSMA-targeted photodynamic therapy by harvesting tissue-penetrating properties of the NIR light, we developed a bacteriochlorophyll-based PSMA-targeted photosensitizer (BPP), consisting of three building blocks: (1) a PSMA-affinity ligand, (2) a peptide linker to prolong plasma circulation time and (3) a bacteriochlorophyll photosensitizer for NIR fluorescence imaging and photodynamic therapy (Qy absorption maximum at 750 nm). BPP exhibited excellent PSMA-targeting selectivity in both subcutaneous and orthotopic mouse models. The nine D-peptide linker in BPP structure prolonged its plasma circulation time (12.65 h). Favorable pharmacokinetic properties combined with excellent targeting selectivity enabled effective BPP tumor accumulation, which led to effective PDT in a subcutaneous prostate adenocarcinoma mouse model. Overall, bright NIR fluorescence of BPP enables effective image guidance for surgical resection, while the combination of its targeting capabilities and PDT activity allows for potent and precise image-guided photodynamic treatment of PSMA-expressing tumors.     

PHOTODYNAMIC EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE UPTAKE OF RHODAMINE 123 BY MITOCHONDRIA OF INTACT MURINE L929 FIBROBLASTS AND CHINESE HAMSTER OVARY Kl CELLS

Abstract— It was shown that the cationic fluorescence probe rhodamine 123 accumulates in mitochondria of murine L929 fibroblasts and Chinese hamster ovary Kl epithelial cells due to the driving force of both plasma membrane and mitochondrial membrane potentials. Photodynamic treatment of L929 cells with hematoporphyrin derivative resulted in an increased uptake of rhodamine 123 and a diminished uptake of 1,1,3,3,3',3'-hexamethylindocarbocyanine iodide. This indicates a considerably increased mitochondrial membrane potential, which most likely is the result of a direct or secondary inhibition of the ATP-synthetase, and a decreased plasma membrane potential. The oxygen consumption rate and the ATP level decreased due to photodynamic treatment. Post-incubation of L929 cells subsequent to photodynamic treatment revealed that the uptake of rhodamine 123. the ATP content and the oxygen consumption rate were restored. For all parameters similar results were obtained with CHO-K1 cells, with the exception that during post-incubation the intracellular ATP content remained at the level reached after illumination. These results indicate that photodynamically induced disturbance of mitochondrial functions and the ATP level are not crucial for the loss of clonogenicity of L929 cells. In CHO-K1 cells however, the continuously lowered ATP level may have detrimental consequences for cell survival. The photodynamic stimulation of the rhodamine 123 uptake may be a rather general phenomenon. Because rhodamine 123 exhibits a much higher toxicity towards carcinoma cells than towards other cells, a synergistic interaction between this drug and photodynamic therapy (PDT) may be anticipated, if PDT also stimulates mitochondrial rhodamine 123 accumulation in carcinoma in vivo.     

Length dependence of the coil <--> beta-sheet transition in a membrane environment

The most abundant structural element in protein aggregates is the beta-sheet. Designed peptides that fold into a beta-sheet structure upon binding to lipid membranes are useful models to elucidate the thermodynamic characteristics of the random coil <-->beta-structure transition. Here, we examine the effect of strand length on the random coil <--> beta-sheet transition of the (KIGAKI)n peptide with the total chain length varying between 7 and 30 amino acids. The beta-sheet content of the peptides in the presence and absence of membranes was measured with circular dichroism spectroscopy. The peptides were titrated with small unilamellar lipid vesicles, and the thermodynamic binding parameters were determined with isothermal titration calorimetry (ITC). Membrane binding includes at least two processes, namely (i) the transfer of the peptide from the aqueous phase to the lipid surface and (ii) the conformational change from a random coil conformation to a beta-sheet structure. CD spectroscopy and ITC analysis demonstrate that beta-sheet formation depends cooperatively on the peptide chain length with a distinct increase in beta-structure for n > 10-12. Binding to the lipid membrane is an entropy-driven process as the binding enthalpy is always endothermic. The contribution of the beta-sheet folding reaction to the overall process was determined with analogues of the KIGAKI repeat where two adjacent amino acids were replaced by their D-enantiomers. The folding reaction for peptides with n >or= 12 is characterized by a negative free folding energy of DeltaG(degree)beta approximately equal -0.15 kcal/mol per amino acid residue. The folding step proper is exothermic with DeltaH(degree)(beta) approximately equal -0.2 to -0.6 kcal/mol per residue and counteracted by a negative entropy term TDeltaS(degree)(beta) = -0.1 to -0.5 kcal/mol per residue, depending on the chain length (18 相似文献   

Photophysics in motionally constrained bioenvironment: interaction of norharmane with bovine serum albumin

Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (DeltaG) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition.     

1,4-二甲基-6,8-二甲氧基-9,10-蒽醌的合成及其与牛血清白蛋白和DNA的相互作用

合成了大黄素类蒽醌衍生物1,4-二甲基-6,8-二甲氧基-9,10-蒽醌(1)并应用紫外光谱、荧光光谱、圆二色谱等方法研究了其与牛血清白蛋白(BSA)和小牛胸腺DNA(ct-DNA)的相互作用.荧光光谱结果表明,化合物1与BSA的相互作用主要以静态猝灭方式使BSA的内源性荧光发生猝灭;圆二色谱表明,化合物1通过疏水作用及形成氢键破坏了α-螺旋结构,导致BSA分子中的α-螺旋含量下降.在pH 7.4时固定DNA的浓度,加入化合物1后,紫外光谱的最大吸收峰发生红移且吸光度加大.荧光光谱表明,化合物1与DNA-4S green NC的结合为竞争性抑制,并可使溶液体系荧光猝灭;圆二色谱表明,随着化合物1的加入,DNA碱基间作用能迅速减弱,表明化合物1与DNA之间为嵌插作用.此外,MTT方法的结果表明,化合物1对结肠癌细胞株HCT116增殖有明显的抑制作用.     

Probing the Binding Interaction of a Phenazinium Dye with Serum Transport Proteins: A Combined Fluorometric and Circular Dichroism Study

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of α-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.     

Effects of acetonitrile on adsorption behavior of bovine serum albumin onto synthetic calcium hydroxyapatite particles

The objective of this study was to examine the effects of acetonitrile (AN) on the adsorption behavior of bovine serum albumin (BSA) onto calcium hydroxyapatite [Ca10(PO4)6(OH)2 Ca10, Hap] materials by combining the ultraviolet (UV) and circular dichroism (CD) measurements of BSA solution. The structural change of BSA molecules with addition of AN was investigated by UV and CD spectroscopy measurements prior to studying adsorption behavior of BSA onto Hap. The CD spectra revealed that the fraction of alpha-helical content of BSA is remarkably decreased at AN concentrations above 30 vol.%, while beta-sheet content is increased. On the other hand, the percentages of random coil and turn contents were decreased only slightly. In addition to this secondary structural change of BSA, the UV spectra suggested that the tertiary structure of protein molecules was also changed by the addition of large amounts of AN; BSA molecules associate to form molecular aggregates at [AN]> or =40 vol.%. From the adsorption of BSA onto Hap particles (ca. 30 nm in the particle length) from a water-AN mixed solution, it was revealed that the adsorption behavior of BSA strongly depends on the change of secondary and tertiary structures of BSA by addition of AN. The contraction of BSA molecules at low AN concentrations (10-20 vol.%) gave their small cross-sectional area, providing a large amount of adsorption (n(BSA)), although n(BSA) was decreased above 30 vol.% AN by enlargement of BSA molecules with solvation and unfolding some alpha-helix domains. The n(BSA) values of the systems with AN exhibited a maximum; n(BSA) was increased at a lower BSA concentration region, although it was decreased at a higher BSA concentration due to self-association. Accompanying the change of n(BSA) with AN addition, the maxima of electrophoretic mobility (em) of the Hap particles were observed for the systems with AN, although the em of Hap particles was normally increased and saturated with increase in protein coverage for the native structure on the system without AN. On the other hand, because the aggregated BSA molecules could be cooperatively bound, the adsorption of BSA onto the Hap particles with large size (108 nm in the particle length) was enhanced in the presence of AN.     

Interaction of a Rhodococcus sp. trehalose lipid biosurfactant with model proteins: thermodynamic and structural changes

One major application of surfactants is to prevent aggregation during various processes of protein manipulation. In this work, a bacterial trehalose lipid (TL) with biosurfactant activity, secreted by Rhodococcus sp., has been identified and purified. The interactions of this glycolipid with selected model proteins have been studied by using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Bovine serum albumin (BSA) and cytochrome c (Cyt-c) have been chosen because of their quite different secondary structures: BSA contains essentially no β-sheets and an average 66% α-helix, whereas Cyt-c possesses up to 25% β-sheets and up to 45% α-helical structure. Differential scanning calorimetry shows that addition of TL to BSA at concentrations below the critical micelle concentration (cmc) shifts the thermal unfolding temperature to higher values. FTIR indicates that TL does not alter the secondary structure of native BSA, but the presence of TL protects the protein toward thermal denaturation, mainly by avoiding formation of β-aggregates. Studies on the intrinsic Trp fluorescence of BSA show that addition of TL to the native protein results in conformational changes. BSA unfolding upon thermal denaturation in the absence of TL makes the Trp residues less accessible to the quencher, as shown by a decrease in the value of Stern-Volmer dynamic quenching constant, whereas denaturation in the presence of the biosurfactant prevents unfolding, in agreement with FTIR results. In the case of Cyt-c, interaction with TL gives rise to a new thermal denaturation transition, as observed by DSC, at temperatures below that of the native protein, therefore facilitating thermal unfolding. Binding of TL to native BSA and Cyt-c, as determined by ITC, suggests a rather nonspecific interaction of the biosurfactant with both proteins. FTIR indicates that TL slightly modifies the secondary structure of native Cyt-c, but protein denaturation in the presence of TL results in a higher proportion of β-aggregates than in its absence (20% vs 3.9%). The study of Trp fluorescence upon TL addition to Cyt-c results in a completely opposite scenario to that described above for BSA. In this case, addition of TL considerably increases the value of the dynamic quenching constant, both in native and denatured protein; that is, the interaction with the glycolipid induces conformational changes which facilitate the exposure of Trp residues to the quencher. Considering the structures of both proteins, it could be derived that the characteristics of TL interactions, either promoting or avoiding thermal unfolding, are highly dependent on the protein secondary structure. Our results also suggest the rather unspecific nature of these interactions. These might well involve protein hydrophobic domains which, being buried into the protein native structures, become exposed upon thermal unfolding.     

Structure, topology, and dynamics of membrane peptides and proteins from solid-state NMR spectroscopy

The high-resolution structure of membrane proteins is notoriously difficult to determine due to the hydrophobic nature of the protein-membrane complexes. Solid-state NMR spectroscopy is a unique and powerful atomic-resolution probe of the structure and dynamics of these important biological molecules. A number of new solid-state NMR methods for determining the depth of insertion, orientation, oligomeric structure, and long-range (10-15 A) distances of membrane proteins are summarized. Membrane protein depths can now be determined using several complementary techniques with varying site-specificity, distance precision, and mobility requirement on the protein. Membrane protein orientation can now be determined with or without macroscopic alignment, the latter providing a novel alternative for orientation determination of intrinsically curvature-inducing proteins. The novel analyses of beta-sheet membrane protein orientation are described. The quaternary structure of membrane peptide assemblies can now be elucidated using a 19F spin diffusion technique that simultaneously yields the oligomeric number and intermolecular distances up to 15 A. Finally, long-range distances up to approximately 10 A can now be measured using 1H spins with an accuracy of better than 1 A. These methods are demonstrated on several beta-sheet membrane peptides with antimicrobial activities and on two alpha-helical ion-channel proteins. Finally, we show that the nearly ubiquitous dynamics of membrane proteins can be readily examined using 2D correlation experiments. An intimate appreciation of molecular motion in these systems not only leads to important insights into the specific function of these membrane proteins but also may be exploited for other purposes such as orientation determination.