Effect Of GdnHCl Research Articles

Denaturant-Mediated Modulation of the Formation and Drug Encapsulation Responses of Gold Nanoparticles.

The extensive and diversified applications of the well-known plasmonic nanoparticle systems along with their easy and environment-friendly synthesis strategies drive us to investigate in-depth this important research field. In the current scenario, our present study deals with an important plasmonic nanomaterial, i.e., globular protein, and human serum albumin (HSA)-conjugated gold nanoparticle (HSA-Au NP) system. The well-known chemical denaturants, urea and guanidine hydrochloride (GdnHCl or GnHCl), are investigated to show detrimental effects toward the formation of gold nanoparticles; however, the effect of GdnHCl is observed to be much prominent compared to that of urea. The synthesized nanoparticle system is found to be highly biocompatible from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cytotoxicity assay, and therefore, the applications of encapsulation of the well-known anticancer drug molecule, doxorubicin hydrochloride (Dox), in the nanoparticle system are further studied. In this drug encapsulation study, drug-metal complexation between Dox and HAuCl4·3H2O has been discussed elaborately. Similar to the nanoparticle formation, the effects of denaturants on drug encapsulation have also been discovered, and interestingly, it has been observed that urea plays a positive role, whereas GdnHCl plays a negative or detrimental role toward drug encapsulation in the synthesized gold nanoparticle system. The detailed photophysical mechanisms behind the drug encapsulation in the synthesized plasmonic nanosystem at every stage have also been explored. Overall, this study will conclusively explain the influences of the extensively used chemical denaturants on the synthesis and drug encapsulation behaviors of a well-known protein-conjugated gold nanoparticle, and as a consequence, it can be highly useful and acceptable to the biomedical and pharmaceutical research communities.

Fluorescence quenching, structural and unfolding studies of a purified cysteine protease, ZCPG from Zingiber montanum rhizome

A first attempt was made to study the fluorescence quenching, structure and unfolding nature of the purified Zingiber montanum (J.Koenig) Link ex A.Dietr. cysteine protease glycoprotein (ZCPG). ATR-IR spectra showed the presences of amide groups along with carbohydrate stretch indicating the glycoprotein nature. UV–vis spectra determined the presences of peptide groups and aromatic sidechains of tyrosine, tryptophan and phenylalanine. Far UV-Circular Dichroism spectrum revealed that the secondary structure consists of 47.6% α-helix, 14.1% β-sheet, 16.1% β-turn, and 22.2% random coil. CD signals revealed pronounced structural stability until 70°C followed by a significant variation in the secondary structure content in the transition temperature between 80–90°C. ZCPG retained most of its secondary structure in the pH range of 3.0–10.0. The extrinsic study shows that at pH 2.0, ZCPG revealed characteristics of a molten globule-like state exhibiting strong ANS binding. The effect of GdnHCl on ZCPG evaluated by far-CD emission maximum and fluorescence emission revealed that the unfolding was incomplete determining the stability of the protein. The microenvironment of the tryptophan residues indicated the presence of relatively exposed single tryptophan residue (per monomer) with positively charged side chains.

A computational integrating kinetic study on the flexible active site of human acetaldehyde dehydrogenase 1

In order to gain more insight into the relation between the structure of acetaldehyde dehydrogenase 1 (ALDH1) and its catalytic and regional active site properties, the denaturant guanidine hydrochloride (GdnHCl) was employed in this study. The effects of GdnHCl on ALDH1 conformational and functional changes were evaluated by kinetic analysis and by performing computational molecular dynamics (MD) simulations. We found that direct binding of GdnHCl to ALDH1 induced complete inactivation prior to conspicuous changes in its tertiary structure or hydrophobic exposure, indicating that the active site is flexible compared to the overall structure. Kinetic experimental results and computational simulations indicated that there are specific sites on ALDH1 to which guanidine binds, resulting in blocking of catalytic function without a large degree of structural disruption. These sites may lay specifically in a cofactor-binding region, which was suggested by the observation of mixed-type inhibition. Our study provides insight into the flexibility of the ALDH1 active site through the use of GdnHCl denaturant and computational simulations to suggest possible binding mechanisms of inhibitors for the clinical applications.

Spectroscopic Probing of the Unfolding of the CopC Induced by Surfactant.

In this work, results of studies on the interactions of CopC and its mutants with the cationic surfactant Cetyltrimethyl Ammonium Bromide (CTAB) monitored by fluorescence spectroscopy, far- UV circular dichroism (CD) and fluorescence lifetime have been presented and discussed. The interaction of CTAB with CopC leads to stabilize a second intermediate, which is considered that the structure of C-terminal was unfolded completely and the structure of the N-terminal was partially unfolded. Notably, GdnHCl leads to refolding of the second intermediate and form the first intermediate, which has also been found on unfolding of CopC by sodium dodecyl sulfate (SDS). The refolding of a protein by GdnHCl in the presence of CTAB was reported. The results suggested that the formation of micelle-like aggregates interaction with the protein results in formation of intermediates of apoCopC and the effect of GdnHCl on micelle-like aggregates result in the folding of the second intermediate.

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50% methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0-3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0-4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k unf of native protein to be 5.407 × 10(-5) s(-1) and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.

Thermodynamic Characterization of the Osmolyte Effect on Protein Stability and the Effect of GdnHCl on the Protein Denatured State

To understand the biomolecular interactions of osmolytes or guanidine hydrochloride (GdnHCl) with protein functional groups, we have determined the apparent transfer free energies (Delta'(tr)) of a homologous series of cyclic dipeptides (CDs) from water to aqueous solutions of osmolytes or GdnHCl through solubility measurements, as a function of osmolyte or GdnHCl concentration at 25 degrees C under atmospheric pressure. The materials investigated in the present study included the CDs of cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cyclo(Val-Val), the osmolytes of trimethylamine N-oxide (TMAO), sarcosine, betaine, proline, and sucrose, and the denaturant of GdnHCl. We observed positive values of (Delta'(tr)) for CDs from water to osmolyte, indicating that interactions between osmolytes and CDs are unfavorable. In contrast, negative (Delta'(tr)) contributions were observed for CDs from water to GdnHCl, revealing that favorable interactions are predominant. The experimental results were further used to estimate the transfer free energies (Delta'(tr)) of the peptide bond (-CONH-), the peptide backbone unit (-CH2C=ONH-), and various functional groups from water to aqueous solutions of osmolyte or GdnHCl.

Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein

The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the inhibitor protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500–650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the inhibitor protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of inhibitor protein, indicating that the protein stabilizes the global structure of particulate F1.

Structural alterations and inhibition of unisite and multisite ATP hydrolysis in soluble mitochondrial F1 by guanidinium chloride.

The effect of guanidinium chloride (GdnHCl) on the ATPase activity and structure of soluble mitochondrial F1 was studied. At high ATP concentrations, hydrolysis is carried by the three catalytic sites of F1; this reaction was strongly inhibited by GdnHCl concentrations of <50 mM. With substoichiometric ATP concentrations, hydrolysis is catalyzed exclusively by the site with the highest affinity. Under these conditions, ATP binding and hydrolysis took place with GdnHCl concentrations of >100 mM; albeit at the latter concentration, the rate of hydrolysis of bound ATP was lower. Similar results were obtained with urea, although nearly 10-fold higher concentrations were required to inhibit multisite hydrolysis. GdnHCl inhibited multisite ATPase activity by diminishing the V(max) of the reaction without significant alterations of the Km for MgATP. GdnHCl prevented the effect of excess ATP on hydrolysis of ATP that was already bound to the high-affinity catalytic site. With and without 100 mM GdnHCl and 100 microM [3H]ATP in the medium, F1 bound 1.6 and 2 adenine nucleotides per F1, respectively. The effect of GdnHCl on some structural features of F1 was also examined. GdnHCl at concentrations that inhibit multisite ATP hydrolysis did not affect the exposure of the cysteines of F1, nor its intrinsic fluorescence. With 100 mM GdnHCl, a concentration at which unisite ATP hydrolysis was still observed, 0.7 cysteine per F1 became solvent-exposed and small changes in its intrinsic fluorescence of F1 were detected. GdnHCl concentrations on the order of 500 mM were required to induce important decreases in intrinsic fluorescence. These changes accompanied inhibition of unisite ATP hydrolysis. The overall data indicate that increasing concentrations of GdnHCl bring about distinct and sequential alterations in the function and structure of F1. With respect to the function of F1, the results show that at low GdnHCl concentrations, only the high-affinity site expresses catalytic activity, and that inhibition of multisite catalysis is due to alterations in the transmission of events between catalytic sites.

Conformational changes at the active site of bovine pancreatic RNase A at low concentrations of guanidine hydrochloride probed by pyridoxal 5′-phosphate

The α-amino group of Lys-1 and the ϵ-amino groups of Lys-41 and Lys-7 were labeled with pyridoxal 5′-phosphate (PLP) separately. The effects of GdnHCl on the activities and the fluorescence properties of the derivatives were compared. Both the fluorescence intensity and anisotropy of the probe introduced at the active site Lys-41 decreased obviously during denaturation by low concentrations of GdnHCl indicating conformational change of the active site is a parallel event to the inactivation of the enzyme. Time-correlated fluorescence lifetime measurements revealed the existence of two conformational states of PLP-modified RNase A and a shift of the conformation in favor of the slow-decay component with increasing GdnHCl concentration. The decrease in activity of RNase A at low concentrations of GdnHCl is therefore due to partial unfolding of the molecule, particularly at the active site. The experimental results of this work support the suggestion that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole.

Comparison of the Conformational Stability of the Molten Globule and Native States of Horse Cytochrome c: Effects of Acetylation, Heat, Urea and Guanidine-Hydrochloride

The molten globule state has been assumed to be a major intermediate of protein folding. We compared the stability of the native and acidic molten globule states of horse ferricytochrome c against heat, urea and guanidine hydrochloride (Gdn-HCl) using the intact species and species modified by various degrees of acetylation of the lysyl ε-amino groups. After acetylation, the amino groups cannot protonate at acidic pH. Thermal and urea-induced unfolding transitions measured by far-UV circular dichroism and differential scanning calorimetry showed that, whereas acetylation stabilizes the molten globule state at pH 2, it destabilizes the native state at pH 7, suggesting a difference in their mechanisms of conformational stability. On the other hand, the effects of Gdn-HCl were remarkable. Contrary to what was expected from the thermal and urea-induced unfolding transitions, the Gdn-HCl-induced unfolding transition of the native state at pH 7 was insensitive to the extent of acetylation. At pH 2, Gdn-HCl at low concentrations stabilized the molten globule state and, at high concentrations, destabilized it. Consideration of the difference in the effects of Gdn-HCl from those of urea or heat indicated that, whereas the net positive charge repulsion destabilizes the molten globule state at pH 2, the local negative charge repulsion produced by acetylation of amino groups, and not the net charge, critically destabilizes the native state at pH 7. These results predict that, because of its ionic nature, Gdn-HCl will produce substantially different effects on the conformational states of some proteins compared with those of urea.