Detailed circular dichroism and fluorescence studies at different pHs have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc). The change in CD in the 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that in the 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. The results suggested that melting of the tertiary structure to a pre-molten globule form takes place at 45 degrees C, which is much lower than the temperature (T(m) = 74 degrees C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pK(a) of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.5. A 'high temperature' unfolding phase was observed which was, however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol), indicating that the 'high temperature' form is possibly stabilized due to interhelical disulfide bonds. Depletion of Ca(2+) ions resulted in a marked decrease in the stability of the secondary structure of the enzyme.

protein folding ͉ conformational dynamics ͉ diffusion coefficient ͉ unfolded state T he intestinal fatty acid binding protein (IFABP) belongs to a family of proteins that bind a variety of ligands (fatty acids, bile salts, and retinoids) into a large cavity located in the interior of the protein. The structure has been determined by both x-ray (1, 2) and NMR methods (3, 4). The x-ray structure shown in Fig. 1 is believed to represent the closed form of the apo protein, whereas the NMR structure suggests the open form (4). The structure of IFABP consists of 2 ␤-sheets, each containing 5 ␤-strands, and a small helical region. This protein has been an excellent model system for folding studies of ␤-sheet proteins because it is small (15 kDa), monomeric, and does not contain any proline or cysteine residues. Equilibrium unfolding transitions of IFABP monitored by steady state fluorescence or CD measurements can be fit by typical two-state model. However, two recent kinetic studies (5, 6) indicated the presence of intermediates, the first one forming within 200 sec. A second intermediate, formed with a rate constant of Ϸ2,000 sec Ϫ1 , contains the majority of the secondary structure (6). Previous 19 F NMR studies as a function of urea concentration had indicated the presence of an intermediate state in addition to the folded and unfolded forms (7). Hodsdon and Frieden (8), monitoring urea denaturation by NMR, found several unidentified resonances in heteronuclear sequential quantum correlation (HSQC) spectra at low urea concentrations, which disappeared at higher urea concentrations, again indicating the presence of intermediates. In the present study, we have explored the possibility of using fluorescence correlation spectroscopy (FCS) as a tool to measure the conformational dynamics and diffusional properties of apo IFABP in its folded, unfolded, and intermediate states.FCS is emerging as an important technique in chemistry, biophysics, and biochemistry for its applications in measuring diffusional properties and chemical kinetics at low molar concentrations (9, 10). The technique involves measuring fluorescence fluctuations resulting from the changes in the number of fluorophores due to diffusion or chemical reaction under conditions of thermodynamic equilibrium in a small observation volume.There are generally two kinds of applications of FCS in protein biophysics. First, the diffusion time and hence the diffusion coefficient of a protein can be measured very accurately. Second, by suitably optimizing the measurement conditions, FCS can be used to study protein dynamics or conformational events in the microsecond time scale (11).In earlier studies, we mutated specific residues of IFABP to cysteine and examined the properties of the mutated proteins (12) as well as proteins for which the fluorescent probe fluorescein was covalently attached to the cysteine residue (13). These studies showed that the probe attached to cysteine at position 60 (V60C) was located within the interior cavity and that the fluorescence p...

The simplest dynamic model for an unfolded protein is a statistical coil that continually undergoes substantial conformational fluctuations. A growing number of studies indicate that the unfolded protein is not a simple random coil but rather forms transient structures. We have directly measured the rate of conformational fluctuations of unfolded intestinal fatty acid binding protein (131 aa, 15 kDa) by using fluorescence self-quenching in combination with fluorescence correlation spectroscopy. The conformational fluctuations in this state have an apparent relaxation time, R, of 1.6 sec in 3 M guanidine-HCl at pH 7 and 20°C. The value of R increases with increasing solution viscosity, suggesting a diffusive process. In the molten globule state at pH 2, R is 2.5 sec, increasing further with the formation of salt-induced secondary structure. These measurements, which should be widely applicable to other systems, can provide important information about the still incompletely understood conformational properties of unfolded proteins and the mechanism of protein folding. Little is known, however, about the dynamics between different conformers in the unfolded state. Probing these conformational transitions is not trivial for two reasons. First, the dynamics are expected to be rapid (sec) and inaccessible to NMR and other commonly used experimental techniques. Second, the difference in spectroscopic signatures between two conformational states is small or even absent, thus making it difficult to find a suitable probe. In this study, we have used fluorescence self-quenching of tetramethyl rhodamine (TMR) as analyzed by fluorescence correlation spectroscopy (FCS) to study the dynamics of the unfolded state under different solution conditions.To perform these experiments we have incorporated a fluorescent probe in two positions of the intestinal fatty acid binding protein (IFABP), 48 residues apart, and have directly measured both the rate of diffusion of the whole molecule and the rate of internal dynamics of the chain in the unfolded state. IFABP consists of two ␤-sheets enclosing a large cavity into which the fatty acid binds (3). The protein (131 residues, 15 kDa) does not contain either cysteine or proline residues, but numerous mutations of residues to cysteine, to which fluorescence probes have been covalently attached (4) have been made with few deleterious effects. Thus, this protein provides an excellent model system for these studies. The persistence of structure in the unfolded state of IFABP was observed by Hodsdon and Frieden (5). In the present study two polar residues, in the two different ␤-sheets, have been mutated to cysteine (D59C͞E107C) and then modified with TMR-5-maleimide. Two other mutants were generated, each with only one residue replaced by cysteine and modified by TMR (D59C-TMR and E107C-TMR). Relative to the labeled single mutants, the doubly labeled mutant is significantly quenched in the presence of 2 M guanidine-HCl (Gdm⅐HCl). We conclude that this quenching (fluorescence self-quenching) ...

We report here a facile and green synthetic approach to prepare magnetite (Fe(3)O(4)) nanoparticles (NPs) with magnetic core and polyethylene glycol (PEG) surface coating. The interaction of the bare and PEG-coated Fe(3)O(4) NPs with cytochrome c (cyt c, an important protein with direct role in the electron transfer chain) is also reported in this study. With ultrasonication as the only peptization method and water as the synthesis medium, this method is easy, fast, and environmentally benign. The PEG coated NPs are highly water dispersible and stable. The bare NPs have considerable magnetism at room temperature; surface modification by PEG has resulted in softening the magnetization. This approach can very well be applicable to prepare biocompatible, surface-modified soft magnetic materials, which may offer enormous utility in the field of biomedical research. Detailed characterizations including XRD, FTIR, TG/DTA, TEM, and VSM of the PEG-coated Fe(3)O(4) NPs were carried out in order to ensure the future applicability of this method. Although the interaction of bare NPs with cyt c shows reduction of the protein, efficient surface modification by PEG prevents its reduction.

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.