Protein Conformation Changes Induced by a Novel Organophosphate

Sep 12, 2007 - Mingming Zhen , Junpeng Zheng , Lei Ye , Shumu Li , Chan Jin , Kai Li .... Xiu-feng Zhang , Lei Chen , Qian-fan Yang , Qian Li , Xiao-r...
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J. Phys. Chem. C 2007, 111, 14327-14333

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Protein Conformation Changes Induced by a Novel Organophosphate-Containing Water-Soluble Derivative of a C60 Fullerene Nanoparticle Xiu-feng Zhang,† Chun-ying Shu,‡ Ling Xie,† Chun-ru Wang,‡ Ya-zhou Zhang,† Jun-feng Xiang,† Lin Li,† and Ya-lin Tang*,† Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Key Laboratory of Molecular Nanostructures and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China ReceiVed: April 28, 2007; In Final Form: July 2, 2007

Water-soluble fullerene derivatives have attracted great attention in biological and medical applications. In particular, for any potential in vivo application, the interaction of water-soluble fullerene nanoparticles with human serum albumin (HSA) is crucial. In this study, we synthesized a novel organophosphate-containing water-soluble derivative of C60 (C60Om(OH)n(C(PO3Et2)2)l (m ≈ 8, n ≈ 12, and l ≈ 1), abbreviated as TEMDPCF). To explore the influence of an organophosphate-containing water-soluble derivative of C60 nanoparticles on the conformational changes of HSA, we have investigated the interaction of TEMDP-CF with HSA by biophysical methods, mainly 31P NMR, MALDI-TOF mass spectroscopy, fluorescence, fluorescence dynamics, UV spectroscopy, FT-IR, and CD, for the first time. 31P NMR and MALDI-TOF MS analysis have proven the formation of the HSA-TEMDP-CF complex, which is further confirmed by fluorescence and fluorescence dynamics results. We observed a quenching of fluorescence of HSA in the presence of TEMDP-CF and also analyzed the quenching results using the modified Stern-Volmer equation, and a red shift in the emission maximum wavelength can be explained as the result of changes in the ternary structure near the binding site. From fluorescence, fluorescence dynamics, and energy transfer experiment parameters, we can predict the possible binding position of TEMDP-CF on the HSA at the site of subdomain IIA, which also agrees with the reported literature. Most significantly, the percentage of the HSA R-helix and β-sheet structure increased, and the β-turn structure decreased in the CD and FTIR analysis results, revealing that the protein becomes more compact upon association with TEMDP-CF. Furthermore, the increase of the R-helix amount, at least on the structure of HSA, may ascribe to the distinct property of the water-soluble TEMDP-CF nanoparticles.

Introduction Fullerenes and their derivatives1 have attracted great attention in biological and medical applications. Fullerenes, particularly those derivatized to allow water solubility, are known to exhibit a variety of pharmacological activities such as antiviral and antibacterial properties, antioxidant and neuroprotective activities, and cell signaling and apoptosis and have the potential to be developed as enzyme inhibitors and diagnostic agents.2 Fullerenes and their derivatives can generate singlet oxygen under visible light and exhibit a suppressive effect on tumor tissue without any damage to normal skin.3 Water-soluble fullerene derivatives have not shown acute toxicity after oral administration in rats,4 and they have different affinities in vivo according to their diversity of water-soluble groups. Organic phosphorus compounds are biologically active species,5 owing to their high affinity to bone surfaces, and organophosphates have been widely used in medical treatments of bone disease6 during the last 20 years. In this paper, a novel organophosphatecontaining water-soluble derivative of C60 (C60Om(OH)n(C(PO3 Et2)2)l (m ≈ 8, n ≈ 12, and l ≈ 1), abbreviated as TEMDP-CF) nanoparticles was synthesized. The addition of phosphate-based * Corresponding author. Tel.: +86/10/62522090; fax: +86/10/62522090; e-mail: [email protected]. † State Key Laboratory for Structural Chemistry of Unstable and Stable Species. ‡ Key Laboratory of Molecular Nanostructures and Nanotechnology.

substituents to the fullerene cage may provide potential applications in the medical treatment of bone diagnosis. Human serum albumin (HSA), the most abundant protein in serum, is the most important drug carrier protein.7 HSA aids in the transport, distribution, and metabolism of many endogenous and exogenous ligands, including fatty acids, amino acids, metal ions, and numerous pharmaceuticals, and contributes significantly to colloid osmotic blood pressure.8 Almost every pharmaceutical compound injected in the blood finds itself in the presence of a high concentration of HSA, known to have a strong affinity for a variety of chemical species. Because of its clinical and pharmaceutical importance, much attention has therefore been paid to investigating the interactions of a number of natural and synthetic ligands with this protein.9 The growing interest in fullerenes provides a compelling reason to investigate their interaction with proteins. There are some reports about the interaction of fullerene derivatives and HIV protease,10 fullerene-specific antibodies,11 and the interaction of hydrated C60 fullerene with HSA using DSC and ESR spin-labeling techniques.12 A stable HSA-fullerene complex was synthesized, purified by size-exclusion chromatography, and characterized.13 Further investigation of the free radical scavenger activity of the HSA-fullerene complex indicated that the antioxidant capabilities of the HSA protein were almost unaffected. As we know, the conformational changes of a protein are associated with its biological function, folding, stability, and

10.1021/jp073267u CCC: $37.00 © 2007 American Chemical Society Published on Web 09/12/2007

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intracellular transport. To our best knowledge, however, there has been no study probing the protein conformation change resulting from interactions between the protein amino acids and the fullerene derivatives. In particular, for any potential in vivo application, the interaction of water-soluble fullerene nanoparticles with a natural protein such as HSA is crucial. To explore the influence of organophosphate-containing water-soluble derivatives of C60 nanoparticles on the conformational structure of HSA, in this study, we have explored the interaction of TEMDP-CF with HSA by biophysical methods, mainly 31P NMR, MALDI-TOF mass spectroscopy, fluorescence, fluorescence dynamics, UV spectroscopy, FT-IR, and CD spectroscopy. This study may provide valuable answers to the growing concern regarding the effects of water-soluble fullerene nanoparticles on the changes of secondary and tertiary structures in HSA conformation. Experimental Procedures Materials. HSA, 96-99% purity (catalog no. A-1653), was purchased from Sigma and used without further purification. C60 was produced by the standard method.14 Analytical grade tetraethyl methylenediphosphonate, NaH, and toluene were purchased from Sigma. To prepare the phosphate buffered solution (PBS, pH 7.4), analytical grade NaH2PO4, Na2HPO4, and NaCl were used as received, and doubly distilled water was used as the solvent throughout the experiments. Preparation and Characterization of TEMDP-CF. The organophosphate-containing water-soluble derivative of C60 (TEMDP-CF) was synthesized by an improved Bingel method.15 Briefly, NaH (1 g) was added to a solution of C60 (5 mg) and tetraethyl methylenediphosphonate (10 µL) in dry toluene (25 mL) at room temperature. After stirring for 1 h, deionized water (50 mL) was added dropwise to the previous mixture. The resulting solution was stirred at room temperature for additional 24 h, and the organic layer became colorless (i) NaH/toluene

C60 + CH2(PO3Et2)2 9 8 C60Om(OH)n(C(PO3Et2)2)l (ii) H O 2

The brown aqueous layer was then separated from the colorless organic layer and concentrated. This residue was purified on a Sephadex G-25 (Pharmacia) size-exclusion gel column with distilled water as the eluent. The brown fraction collected (pH ) 6 to ∼7) was concentrated in a vacuum. The overall reaction yield was more than 80%. The molecular composition was determined by MALDI-TOF mass spectroscopy, FT-IR, 31P NMR, and XPS (details are in the Supporting Information), and the results showed that the molecular formula of the synthesized C60 derivative is (C60Om(OH)n(C(PO3 Et2)2)l (m ≈ 8, n ≈ 12, and l ≈ 1) with a molecular weight of 1338. The possible molecular structure of TEMDP-CF is shown in Figure 1a. Fluorescence, Fluorescence Dynamics, NMR, MALDITOF, CD, and FT-IR Spectroscopy. Fresh stock solutions of TEMDP-CF and HSA were prepared by being dissolved in PBS. The measured sample was prepared by mixing a quantity of TEMDP-CF with the HSA solution and then was diluted by PBS. The sample solutions were kept at room temperature (25 °C) for more than 12 h before the experiments were performed. Fluorescence spectra were recorded on a Hitachi F4500 spectrofluorometer in a 1 cm path-length quartz cell using an excitation wavelength of 295 nm. Slit widths (5 nm), scan speed (240 nm/min), and excitation voltage (400 V) were kept constant within each data set. Excitaion and emission bandwidths were 5 nm. Fluorescence dynamics measurements were carried out

Figure 1. Schematic representation of (a) TEMDP-CF and (b) TEMDP-CF binding site in HSA’s subdomain IIA (PDB entry 1AO6).

at the ambient temperature of 25 °C on a FLS920 time-resolved spectrofluorometer with a single-photon counting system (Edinburgh Analytical Instruments). 31P NMR spectra were measured on a Bruker Avance 600 NMR at 300 K. The matrixassisted laser desorption and ionization time-of-flight (MALDITOF) mass spectrometry was performed to analyze the samples’ composition with 4-hydroxy-R-cyano cinnamic acid as the matrix. XPS measurements were taken with an ESCALab220iXL electron spectrometer from VG Scientific using 300 W Al KR radiation. CD measurements were carried out on a Jasco815 automatic recording spectropolarimeter in a cell with a 1 mm path-length at 25 °C. Spectra were collected at a scan speed of 500 nm/min and response time of 1 s. Each spectrum was the average of four scans and was corrected by the PBS solution. Solution pH values were measured with a PHS-3C instrument. Infrared spectra were collected at room temperature with a Nicolet Nexus 670 FT-IR spectrometer equipped with a zinc selenide (ZnSe) attenuated total reflectance (ATR) accessory, a deuterated triglycine sulfate (DTGS) detector, and a KBr beam splitter. A 2048 scan water vapor spectrum was recorded previously for automatic correction during data collection. After an open background spectrum was recorded, the sample solution was spread on the ZnSe wafer, and its 256 scan spectrum was collected at a resolution of 4 cm-1. The protein concentration was 40 mM, and the molar ratios of drug to protein (Cdrug/CHSA) were 0.5 and 1.0, respectively. According to the previous procedures, the infrared spectra of the free protein, drug-protein mixture, and drug at corresponding concentrations were collected, respectively. Subtraction was performed between the spectrum of HSA in buffer solution and that of the buffer solution to obtain the free protein spectrum with the subtraction criterion that the original spectrum of the protein solution at about 3600 cm-1 and in the band between 2200 and 1800 cm-1 was featureless.16 The protein spectra after interacting with the drug were obtained by subtracting the spectra of the protein and drug solution from those of the drug in corresponding concentrations. Baseline corrections were carried out in the range of 1700-1600 cm-1 to obtain the amide I band. The number,

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Figure 2. 31P NMR spectra of (a) 40 µM TEMDP-CF and (b) 40 µM TEMDP-CF in the presence of 40 µM HSA.

position, and width of the component bands were estimated by performing Fourier self-deconvolution and a second derivative to the protein infrared amide I band. On the basis of these parameters, a curve-fitting process was carried out by Galactic Peaksolve software (version 1.0) to obtain the best Gaussianshaped curves that fit the original protein spectra. After the individual bands with representative secondary structures were identified, the percentages of each secondary structure of HSA were calculated using the area of their respective component bands. Results and Discussion Interaction of TEMDP-CF with HSA. NMR has been extensively used as a useful method for obtaining information on the interactions between macromolecules and small ligand molecules.17 The 31P NMR spectra of TEMDP-CF with and without HSA are shown in the Figure 2. The 31P NMR spectrum exhibits a single peak at δ 18.08, indicating the existence of only one phosphorus environment, which is a signal from the tetraethyl methylenediphosphonate of TEMDP-CF. It is obvious that a new signal peak with a chemical shift at δ 21.6 appears and that the chemical shift at δ 18.4 is broadened in the presence of HSA. The chemical shift change of 31P of TEMDP-CF in the presence of HSA could be induced by HSA-TEMDP-CF interactions, which could be attributed to the HSA-TEMDPCF complex. Since the HSA molecule is much larger and less mobile than that of TEMDP-CF, the mobility of the bound form is reduced dramatically as compared to that of the free form, and the line broadening at δ 18.4 could be explained by assuming a rapid exchange of TEMDP-CF molecules between the bound and the free forms when TEMDP-CF is close to HSA. It is noted that few 31P NMR spectra are applied in the study of drug-protein interactions. 31P NMR has some advantages over 1H NMR and 13C NMR. Because 31P is the only naturally occurring P isotope (100% natural abundance), all P species within a sample can potentially be detected by NMR spectroscopy. P gives a simple signal peak that makes the signals less overlapped and the interpretation of observed spectra relatively easy. In addition, there is no background 31P signal in water, and solvent suppression is unnecessary. The addition of an organophosphate group to the C60 cage makes the fullerene detectable by 31P NMR. The changes of the P chemical shift due to the bond to the protein may provide a potential probe in further understanding the interaction of fullerene and biological macromolecules.

Figure 3. Negative-ion MALDI-TOF mass spectra of (a) HSATEMDP-CF complex and (b) monomeric HSA.

Figure 3 shows MALDI-TOF MS for HSA-TEMDP-CF with a molecular mass of 68 021 and HSA with molecular mass of 66 681. The MALDI-TOF mass spectra analysis unambiguously exhibits that the molecular mass of the HSA-TEMDPCF complex corresponds to a sum of masses of HSA and TEMDP-CF. The molecular mass of TEMDP-CF obtained from this experiment is consistent with the XPS analysis results. However, the MALDI-TOF MS of TEMDP-CF exhibited only a strong C60 ionic peak, while no parent ion peaks were observed similar to other water-soluble fullerenols.18 It is well-known that the laser desorption process easily strips off functional groups from fullerenes.19 Since C60 is completely insoluble in water, the observation of strong C60 ionic signals in water-soluble samples indicates the existence of intact fullerene cages, which is consistent with the modification of C60 to a water-soluble derivative. It seems that the TEMDP-CF molecule in the HSATEMDP-CF complex is stable enough to survive the MALDI conditions. Fluorescence quenching of proteins could be used to retrieve drug-protein binding information.20 When excited at 280 nm, both tryptophan and tyrosine amino acid residues in protein have fluorescence emissions, while at an excitation wavelength of 295 nm, only tryptophan has fluorescence emissions. The fluorescence spectra of HSA in the absence and presence of TEMDP-CF in 20 mM phosphate buffer (pH 7.4) were measured with an excitation wavelength of 295 nm. HSA shows a strong fluorescence emission with a peak at 345 nm with the excitation at 295 nm because of its single tryptophan residue (Trp 214). TEMDP-CF was nonfluorescent under these experimental conditions. The stepwise addition of TEMDP-CF to the HSA solution resulted in considerable quenching of the HSA fluorescence intensity (Figure 4), indicating that TEMDP-CF

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Figure 4. Fluorescence spectra of the HSA--TEMDP-CF system (pH ) 7.4, λex ) 295 nm). (a) 10 µM HSA, (b-l) TEMDP-CF concentration increased from 0 to 55 µM, in the presence of 10 µM HSA, and (m) 55 µM TEMDP-CF.

Figure 5. Modified Stern-Volmer plots of the HSA-TEMDP-CF system (HSA at 10 µM, pH ) 7.4).

interacts with HSA. In addition, a red shift of the wavelength maxima of HSA was observed with the addition of TEMDPCF. It is known that a shift of the maximum emission wavelength corresponded to a polarity change around the chromophore residues. A red shift always indicates that tryptophan residues are, on average, more exposed to the solvent. In this study, the red shift can be explained as the result of changes in the ternary structures of HSA. Binding Mechanism of TEMDP-CF with HSA. Fluorescence quenching was also analyzed using the modified SternVolmer equation21

F0 1 1 + ) F0 - F faKa[Q] fa

(1)

where F0 and F are the relative fluorescence intensities of HSA in the absence and presence of TEMDP-CF, respectively; fa is the fraction of fluorophore accessible to the quencher; [Q] is the concentration of TEMDP-CF; and Ka is the Stern-Volmer quenching constant. From the plots of F0/(F0 - F) versus 1/[Q], the values of fa and Ka were obtained from the values of the intercept and slope, respectively (Figure 5). The value of fa was found to be 1.3, indicating that 76% of the total fluorescence of HSA is accessible to quenchers. The Stern-Volmer quenching constant was found to be 5.6 × 104 M-1. The quenching rate constant of the biomolecule, Kq, was evaluated using the equation

Kq ) Ksv/τ0

(2)

Figure 6. Fluorescence decay curves of 40 µM HSA in the absence (a) and the presence (b) of 40 µM TEMDP-CF. Excitation was performed at λ ) 295 nm, and emission was measured at λ ) 345 nm.

where Ksv is the Stern-Volmer quenching constant (5.6 × 104 M-1), and τ0 is the lifetime of the protein without the quencher. The τ0 value for HSA in the present study was found to be 5.9 ns, and hence, the value of Kq was observed to be 9.5 × 1012 M-1 s-1. The maximum scatter collision quenching constant, Kq, of various quenchers with the biopolymer is 2 × 1010 M-1 s-1. Thus, the rate constant of the protein quenching procedure initiated by TEMDP-CF is greater than the Kq value of the scatter procedure. This indicates that a static quenching mechanism is operative.22 For further insight into the fluorescence quenching processes, the lifetimes of the excited state of trytophan within the protein were measured in the absence and presence of TEMDP-CF at the excitation wavelength at 295 nm, monitored at 345 nm (shown in Figure 6). The fluorescence decay curve of HSA is monoexponential, with a lifetime of 5.9 ns, which is ascribed to the emission of tryptophan within HSA. The decay profiles of the HSA-TEMDP-CF system (HSA/TEMDP-CF ) 1:1) were fitted with a dualexponential function by use of the nonlinear least-squares method with a deconvolution technique, corresponding to lifetimes of 2.0 ns (44%) and 5.9 ns (56%), respectively. The shortened lifetime component (τ ) 2.0 ns) may arise from the bound state of HSA and TEMDP-CF, while the species with a lifetime of 5.9 ns is ascribed to the free state of HSA. Therefore, the fast decay component may arise from the fluorescence quenching of HSA due to the formation of the HSA-TEMDP-CF complex. This finding is also confirmed by the 31P NMR and MALDI-TOF MS analysis results. Taking into account an overlap between the emission spectrum of HSA and the absorption spectrum of TEMDP-CF (Figure 7), an excitation energy transfer mechanism might be assumed. Such a mechanism is not uncommon.23 The importance of energy transfer in biochemistry is that the efficiency of transfer can be used to evaluate the distance r between the ligand and the trytophan residues in the protein. According to Fo¨rster and Sinanoglu’s nonradiative energy transfer theory,24 the efficiency of energy transfer, E, is calculated using the equation

E)1-

R06 F ) 6 F0 R + r 6 0 0

(3)

where F and F0 are the fluorescence intensities of HSA in the presence and absence of TEMDP-CF, r is the distance between

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Figure 7. Overlapping between the fluorescence emission spectra of HSA (a) and the asorption spectra of TEMDP-CF (b).

the acceptor and the donor, and R0 is the critical distance when the transfer efficiency is 49%, which can be calculated by

R06 ) 8.8 × 10-25 K2φJN-4

∑F(λ)(λ)λ4∆λ ∑F(λ)∆λ

TABLE 1: Secondary Structure Determination for Free HSA and the HSA-TEMDP-CF System in pH 7.4 PBS Buffer

(4)

It has been reported for HSA that K2 ) 2/3, φ ) 0.118, and N ) 1.336.25 J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor, given by

J)

Figure 8. CD spectra of the HSA-TEMDP-CF system. (a) 2.5 µM HSA, (b) 2.5 µM HSA + 1.25 µM TEMDP-CF, and (c) 2.5 µM HSA + 2.5 µM TEMDP-CF.

(5)

where F(λ) is the fluorescence intensity of the fluorescent donor in wavelength λ and is dimensionless, and (λ) the molar absorption coefficient of the acceptor in wavelength λ (cm-1 mol-1 L). J can be evaluated by integrating the spectra in Figure 5. Using J ) 1.8 × 10-14, based on these data, we found R0 ) 2.7 nm and r ) 2.7 nm. Thus, the distance between TEMDPCF and Trp residue in HSA is about 2.7 nm. The donoracceptor distance (r < 8 nm)26 indicated that the energy transfer from HSA to TEMDP-CF occurs with a high probability. The distances obtained by this way agree well with literature values of substrate binding to HSA at site IIA.27 C60 fullerene has also been reported to be bound to the subdomain IIA of HSA using time-resolved fluorescence decay experiments, docking calculations, and binding site alignment methods.13,28 From our fluorescence, fluorescence dynamics, and energy transfer experiment parameters, we can predict that the possible binding position of TEMDP-CF on HSA is at site subdomain IIA (shown in Figure 1b). The binding constant of TEMDP-CF is found to be comparable to published values for other organic molecules that strongly bind to the same site of HSA, such as water-soluble antioxidant (-)-epigallocatechin-3-gallate (6.85 × 104 M-1).29 Changes of HSA Conformation Induced by Interaction with TEMDP-CF. Because our 31P NMR, MALDI-TOF- MS, fluorescence, and fluorescence dynamics experiments attempted to confirm the interaction between HSA and TEMDP-CF, it is important to examine how the structure of HSA is affected in the HSA-TEMDP-CF system. When drugs bind to a globular protein, the intramolecular forces responsible for maintaining the secondary and tertiary structures can be altered, resulting in a conformational change of the protein.30 The distinct fluorescence quenching suggested that the HSA-TEMDP-CF combination has changed the microenvironment of HSA. The red shifts of the fluorescence emission wavelengths can be explained as the result of changes in the ternary structure near

amide I components FT-IR results HSA-free TEMDP-CF/HSA ) 0.5 TEMDP-CF/HSA ) 1 CD results HSA-free TEMDP-CF/HSA ) 0.5 TEMDP-CF/HSA ) 1

R-helix (%)

β-sheet (%)

β-turn (%)

59 60 61 54 56 58

25 26 27

16 14 12

the binding site, indicating that the conformation has been changed after protein binding with TEMDP-CF. If the change of protein structure included the transformation of the protein secondary structure in the HSA-TEMDP-CF- complex, it can be reflected in CD spectra and infrared absorption spectra. Figure 8 shows the CD spectra of HSA and the HSATEMDP-CF complex obtained at pH 7.4. The CD spectra of HSA displays two minima in the ultraviolet region, one at 208 nm and the other at 222 nm, which is characteristic of the R-helical structure of a protein. The reasonable explanation is that the negative peaks between 208 and 209 and 222- and 223 nm both contribute to the n f π* transfer for the peptide bond of the R-helix. As is seen in Figure 8, these minima are more pronounced without any significant shift of the peaks in the presence of TEMDP-CF, indicating that the amount of R-helical content in HSA was increased. If the CD result was expressed as MRE (mean residue ellipticity) in deg cm2 dmol-1, the following equation would be used:

MRE ) θobs/(10nlCp)

(6)

where θobs is the CD in millidegrees, n is the number of amino acid residues (585), l is the path length of the cell (1 mm), and Cp is the mole fraction. The helical content was calculated from MRE values at 222 nm using the following equation described in previous literature:31

R-helix ) [(MRE222nm - 2340)/30300] × 100%

(7)

It can be seen in Table 1 that the native HSA solution (2.5 µM) is 54% R-helix, while the R-helix content of HSA increases to 56 and 58% with the molar ratio of TEMDP-CF/protein at 0.5 and 1.0, respectively. Additional evidence regarding the HSA-TEMDP-CF complications comes from infrared spectroscopy results since infrared spectra of proteins exhibit a number of so-called amide

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Zhang et al. component band of the R-helix structure could not be resolved with that of the random coil structure in PBS solution. The free protein contained major amounts of R-helices (55%), β-sheets (22%), β-turn structures (16%), and random coils (6%).33 Table 1 lists the curve-fitted results of protein secondary structure before and after binding with different concentrations of TEMDP-CF. From Table 1, it can be seen that as the percentage of the protein R-helix and β-sheet structure increased, the β-turn structure decreased, which is consistent with CD spectral analysis results. The presence of TEMDP-CF stabilized the HSA structure and increased its ellipticity. It can be seen that the R-helix amount increases and thus the protein becomes more compact upon association with TEMDP-CF. The interaction of TEMDPCF with HSA can weaken the HSA solvation with the water molecules and the self-association within the chain of HSA, so that a much tighter secondary structure of the R-helix becomes dominant in HSA. Meanwhile, this interaction between TEMDPCF and HSA also weakens the hydrogen bonds among TEMDPCF nanoparticles, which is consistent with MALDI-TOF results. It is interesting that the increase of the R-helix amount may ascribe to the distinct property of water-soluble nanoparticles, TEMDP-CF. The present result agrees with those findings that the content of the R-helix of HSA increased in solution containing nanoparticles34 and that HSA becomes more compact upon association with the surfactant molecule.35 To the best of our knowledge, the strong R-helix induced by water-soluble TEMDP-CF nanoparticles, at least on the structure of HSA, is unique to fullerene effects on the protein. The conformational changes of a protein are associated with its biological function, folding, stability, and intracellular transport. Many biological processes have been found to be related to distinct regions, and thus, knowledge of the distinct behaviors of the different secondary structures is of particular interest. Although the occurrence of extensive protein conformation changes in this regime is more or less understood, the structure of the resulting HSA-TEMDP-CF complex needs further investigation. Conclusion

Figure 9. Curve-fit amide I region with secondary structure determination of the HSA-TEMDP-CF complexes at different TEMDP-CF/ HSA ratios in PBS (pH ) 7.4) in the region of 1700-1600 cm-1. TEMDP-CF/HSA (40 µM) ratios of (a) 0:1, (b) 0.5:1, and (c) 1:1.

bands that represent different vibrations of the peptide moiety. Of all the amide modes of the peptide group, the single most widely used one in studies of protein secondary structure is amide I. This vibration mode originates from the CdO stretching vibration of the amide group (coupled to the in-phase bending of the N-H bond and the stretching of the C-N bond) and gives rise to infrared bands in the region between approximately 1600 and 1700 cm-1.32 Figure 9 shows the curve-fitted spectra of the protein infrared amide I bands before and after interaction with TEMDP-CF. The assignments of the component bands for the protein infrared amide I band were as follows: 1648-1660 cm-1 to R-helices and random coil, 1660-1700 cm-1 to β-turn, and 1610-1640 cm-1 to β-sheet structures in PBS buffer.32 The

In this article, a novel organophosphate-containing watersoluble derivative of C60 was synthesized by an improved Bingel method. The molecular composition was determined by MALDITOF mass spectroscopy, FT-IR, 31P NMR, and XPS. The binding of TEMDP-CF with HSA under physiological conditions has been presented for the first time by 31P NMR, MALDITOF MS, fluorescence, fluorescence dynamics, and UV spectroscopycombined with FTIR and CD studies. 31P NMR and MALDI-TOF MS analyses have proven the formation of the HSA-TEMDP-CF complex, which is further confirmed by fluorescence dynamics analysis. The results show that TEMDPCF is a strong quencher of the fluorescence of HSA and binds to the protein with high affinity. The binding of TEMDP-CF to HSA increases the polar environment of the Trp residue, resulting in a red shift in the fluorescence spectra. The binding parameters were calculated using the modified Stern-Volmer equation. The possible binding position of TEMDP-CF on HSA at the subdomain IIA site was deduced from fluorescence, fluorescence dynamics, and energy transfer experiment parameters. The most interesting finding is that as the percentage of the HSA R-helix and β-sheet structure increased, the β-turn structure decreased, and thus, the protein became more compact upon association with TEMDP-CF as shown in the CD and FTIR analysis results.

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