Journal articles: 'Guanidine hydrochloride; Denaturants'

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Stepanenko, Olesya, Olga Stepanenko, Irina Kuznetsova, and Konstantin Turoverov. "The Pathways of the iRFP713 Unfolding Induced by Different Denaturants." International Journal of Molecular Sciences 19, no. 9 (September 15, 2018): 2776. http://dx.doi.org/10.3390/ijms19092776.

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Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome RpBphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH+ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH+ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter A, was found to have a different residual structure in the denaturants used.

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West, S. M., A. D. Guise, and J. B. Chaudhuri. "A Comparison of the Denaturants Urea and Guanidine Hydrochloride on Protein Refolding." Food and Bioproducts Processing 75, no. 1 (March 1997): 50–56. http://dx.doi.org/10.1205/096030897531360.

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Milyaeva, Olga Yu, Richard A. Campbell, Shi-Yow Lin, Giuseppe Loglio, Reinhard Miller, Michail M. Tihonov, Imre Varga, Anna V. Volkova, and Boris A. Noskov. "Synergetic effect of sodium polystyrene sulfonate and guanidine hydrochloride on the surface properties of lysozyme solutions." RSC Advances 5, no. 10 (2015): 7413–22. http://dx.doi.org/10.1039/c4ra14330b.

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A study of the dilational surface viscoelastic properties of mixed solutions of lysozyme and denaturants allows us to characterize the changes of protein tertiary structure in the surface layer upon adsorption at the liquid–gas interface.

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Yang, Ya-Wun, and Chi-Cheng Teng. "Stability of polyomavirus major capsid protein VP1 under denaturants guanidine hydrochloride and urea." International Journal of Biological Macromolecules 22, no. 2 (April 1998): 81–90. http://dx.doi.org/10.1016/s0141-8130(97)00091-3.

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Kujo, Chizu, and Toshihisa Ohshima. "Enzymological Characteristics of the Hyperthermostable NAD-Dependent Glutamate Dehydrogenase from the Archaeon Pyrobaculum islandicum and Effects of Denaturants and Organic Solvents." Applied and Environmental Microbiology 64, no. 6 (June 1, 1998): 2152–57. http://dx.doi.org/10.1128/aem.64.6.2152-2157.1998.

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ABSTRACT NAD-dependent glutamate dehydrogenase (l-glutamate:NAD oxidoreductase, deaminating; EC 1.4.1.2 ) was purified to homogeneity from a crude extract of the continental hyperthermophilic archaeonPyrobaculum islandicum by two successive Red Sepharose CL-4B affinity chromatographies. The enzyme is the most thermostable NAD-dependent dehydrogenase found to date; the activity was not lost after incubation at 100°C for 2 h. The enzyme activity increased linearly with temperature, and the maximum was observed at ca. 90°C. The enzyme has a molecular mass of about 220 kDa and consists of six subunits with identical molecular masses of 36 kDa. The enzyme required NAD as a coenzyme for l-glutamate deamination and was different from the NADP-dependent glutamate dehydrogenase from other hyperthermophiles. The Km values for NAD,l-glutamate, NADH, 2-oxoglutarate, and ammonia were 0.025, 0.17, 0.0050, 0.066, and 9.7 mM, respectively. The enzyme activity was significantly increased by the addition of denaturants such as guanidine hydrochloride and some water-miscible organic solvents such as acetonitrile and tetrahydrofuran. When fluorescence of the enzyme was measured in the presence of guanidine hydrochloride, a significant emission spectrum change and a shift in the maximum were observed but not in the presence of urea. These results indicate that this hyperthermophilic enzyme may have great potential in applications to biosensor and bioreactor processes.

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Gupta, P., A. K. Verma, and P. Chaudhuri (Chattopadhyay). "Investigating the Chaperoning Effect of Nanoparticles in Chemically Denatured zDHFR: An in vitro Study." Asian Journal of Chemistry 33, no. 8 (2021): 1929–34. http://dx.doi.org/10.14233/ajchem.2021.23372.

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Maintenance of native structure and function of the protein is a major concern for industrial production of aggregation prone therapeutically important recombinant proteins. Aggregation may results due to change in the native conformation of proteins under different stress conditions. To overcome the problem of protein aggregation, role of silver and gold nanoparticles have been investigated. The nanoparticles owing to their affirmative interaction with the proteins possess chaperoning activities and protect the native state from denaturation. In the present study, through performing chemical denaturation of zebrafish dihydrofolate reductase using denaturants like guanidine hydrochloride and urea in the presence and absence of gold and silver nanoparticles and monitoring the process through enzyme activity assay and intrinsic tryptophan fluorescence, we have demonstrated the impact of nanoparticles in maintaining native conformation of proteins. Further, the outcome of refolding studies of DHFR protein with nanoparticles monitored by UV-visible spectroscopy was also reported.

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SINGH, R. Rajesh, and Jui-Yoa CHANG. "Investigating conformational stability of bovine pancreatic phospholipase A2: a novel concept in evaluating the contribution of the native-framework of disulphides to the global conformational stability of proteins." Biochemical Journal 377, no. 3 (February 1, 2004): 685–92. http://dx.doi.org/10.1042/bj20030968.

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Bovine pancreatic PLA2 (phospholipase A2) is a 14 kDa protein whose structure is highly cross-linked by seven disulphide bonds. We investigated the structural stability of this enzyme by the method of ‘disulphide-scrambling’ with denaturants such as urea, GdmCl (guanidine hydrochloride), GdmSCN (guanidine thiocyanate) and at high temperatures in the presence of 2-mercaptoethanol (0.2 mM) as thiol initiator. Reverse-phase HPLC was used to follow denaturation. To denature 50% of the native protein, 1.25 M GdmSCN, approx. 3 M GdmCl and higher than 8 M urea were required. Only 20% of the protein was denatured after 2 h at 60 °C, whereas complete denaturation was seen after 2 h at 70 °C and within 30 min at 80 °C. A distinct enhancement of stability was observed when denaturation was conducted in the presence of 10 mM calcium chloride, which has not been reported previously. CD studies of GdmCl denaturation of bovine PLA2 showed that 2.5 M GdmCl was required to denature 50% of the protein in the presence of 0.2 mM 2-mercaptoethanol (in agreement with the HPLC analysis), whereas 6.4 M GdmCl was necessary to denature 50% of the protein in the absence of a thiol initiator. Conformational stability (ΔGwater) was estimated to be 8.7 kcal/mol (1 cal=4.184 J) by ‘disulphide-intact’ denaturation (where ‘native’ disulphide framework was unaffected) and 2.5 kcal/mol by ‘disulphide-scrambling’ denaturation (involved breaking of native disulphides and formation of ‘non-native’ ones). The difference, Δ(ΔGwater), of 6.2 kcal/mol was the conformational stability contributed by the ‘native-framework’ of seven disulphides. Using bovine PLA2 as an example, we have demonstrated a novel comparative technique, where the conformational stability study of a disulphide-containing protein, with a common denaturant, in both the presence and absence of catalytic amounts of a thiol initiator can be used as a convenient method to estimate selectively and quantitatively the actual contribution of the ‘native disulphide bond network’ towards the global conformational stability of the protein.

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FAN, Ying-xin, Ming JU, Jun-mei ZHOU, and Chen-lu TSOU. "Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site." Biochemical Journal 315, no. 1 (April 1, 1996): 97–102. http://dx.doi.org/10.1042/bj3150097.

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It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028–7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulphonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.

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Hovorka,, Štěpán, Vladimír Dohnal,, Ernesto Carrillo-Nava,, and Miguel Costasa. "Infinite dilution activity coefficients for benzene and toluene in water and in aqueous solutions of the protein denaturants urea and guanidine hydrochloride." Journal of Chemical Thermodynamics 32, no. 12 (December 2000): 1683–705. http://dx.doi.org/10.1006/jcht.2000.0706.

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10

Tang, Hong-Min, Wen-Bin Ou, and Hai-Meng Zhou. "Effects of lactic acid and NaCl on creatine kinase from rabbit muscle." Biochemistry and Cell Biology 81, no. 1 (January 1, 2003): 1–7. http://dx.doi.org/10.1139/o02-168.

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The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCl, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding.Key words: lactic acid, creatine kinase, salt-induced, unfolding, molten globule state.

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11

Marsh, E. N., and S. E. Harding. "Methylmalonyl-CoA mutase from Propionibacterium shermanii: characterization of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation." Biochemical Journal 290, no. 2 (March 1, 1993): 551–55. http://dx.doi.org/10.1042/bj2900551.

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A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.

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12

VANHOVE, Marc, Gilliane GUILLAUME, Philippe LEDENT, John H. RICHARDS, Roger H. PAIN, and Jean-Marie FRÈRE. "Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase." Biochemical Journal 321, no. 2 (January 15, 1997): 413–17. http://dx.doi.org/10.1042/bj3210413.

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Class A α-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 α-lactamase, this bond was removed by introducing a Cys-77 → Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N ↔ H ↔ U). Both the folded mutant protein (N) and, to a lesser extent, the thermodynamically stable intermediate, H, were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.

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Watkins, Herschel M., Anna J. Simon, Tobin R. Sosnick, Everett A. Lipman, Rex P. Hjelm, and Kevin W. Plaxco. "Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions." Proceedings of the National Academy of Sciences 112, no. 21 (May 11, 2015): 6631–36. http://dx.doi.org/10.1073/pnas.1418673112.

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Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.

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Smith, Jeni S., and J. Martin Scholtz. "Guanidine Hydrochloride Unfolding of Peptide Helices: Separation of Denaturant and Salt Effects†." Biochemistry 35, no. 22 (January 1996): 7292–97. http://dx.doi.org/10.1021/bi960341i.

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Hu, Qi, Haozhen Hu, Xinyi Zhang, Kyle Fan, Yuning Hong, Colin L. Raston, and Youhong Tang. "In Situ Monitored Vortex Fluidic-Mediated Protein Refolding/Unfolding Using an Aggregation-Induced Emission Bioprobe." Molecules 26, no. 14 (July 14, 2021): 4273. http://dx.doi.org/10.3390/molecules26144273.

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Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for β-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with β-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.

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Kumaran, R., T. Varalakshmi, E. J. Padma Malar, and P. Ramamurthy. "Photophysical Studies on the Interaction of Acridinedione Dyes with Universal Protein Denaturant: Guanidine Hydrochloride." Journal of Fluorescence 20, no. 5 (April 6, 2010): 993–1002. http://dx.doi.org/10.1007/s10895-010-0646-9.

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McDuff, F. O., A. Doucet, and M. Beauregard. "Low concentration of guanidine hydrochloride induces the formation of an aggregation-prone state in α-urease." Biochemistry and Cell Biology 82, no. 2 (April 1, 2004): 305–13. http://dx.doi.org/10.1139/o03-072.

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Canavalia ensiformis (jack bean) α-urease is a hexameric protein characterized by a complex denaturation mechanism. In previous papers, we have shown that a hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANSA) binding conformer could be populated in a moderate concentration of denaturant. This state was obtained under conditions that had no detectable impact on its tertiary structure, as indicated by fluorescence measurements. In the present study, we further characterized this ANSA-binding state in an attempt to understand urease behavior. Evidence presented here shows that the presence of ANSA was not required for the generation of the conformer and that its affinity for ANSA came from an increase in hydrophobicity leading to aggregation. Circular dichroism investigation of urease revealed that it had periodical secondary structure content similar to Klebsiella aerogenes urease (secondary structures calculated on the basis of crystallographic data). The impact of 0.9 M guanidine hydrochloride (GuHCl) on soluble urease secondary structures was minimal but is compatible with a slight increase in beta-sheet structures. Such modification may indicates that aggregation involves amyloid-like fibril formation. Electron microscopy analysis of urease in the absence of GuHCl revealed the presence of urease hexamers (round shape 13 nm in diameter). These particles disappeared in the presence of moderate denaturant concentration owing to the formation of aggregates and fibril-like structures. The fibrils obtained in 1.5 M GuHCl had an average diameter of 6.5 nm, suggesting that urease hexamers dissociated into smaller oligomeric forms when forming such fibrils.Key words: protein structure, protein folding, denaturation, aggregation, multimeric proteins, protein fibrils, hydrophobicity, molten globule state.

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DOSS-PEPE, ELLEN W., ERICA L. CAREW, and JANE F. KORETZ. "Studies of the Denaturation Patterns of Bovine Alpha-Crystallin Using an Ionic Denaturant, Guanidine Hydrochloride and a Non-Ionic Denaturant, Urea." Experimental Eye Research 67, no. 6 (December 1998): 657–79. http://dx.doi.org/10.1006/exer.1998.0561.

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19

Basak, Sujit, R. Paul Nobrega, Davide Tavella, Laura M. Deveau, Nobuyasu Koga, Rie Tatsumi-Koga, David Baker, Francesca Massi, and C. Robert Matthews. "Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed βα protein." Proceedings of the National Academy of Sciences 116, no. 14 (March 15, 2019): 6806–11. http://dx.doi.org/10.1073/pnas.1818744116.

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The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

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Shimizu, Akio, Koichi Fumino, Kenichi Yukiyasu, and Yoshihiro Taniguchi. "NMR studies on dynamic behavior of water molecule in aqueous denaturant solutions at 25 °C: Effects of guanidine hydrochloride, urea and alkylated ureas." Journal of Molecular Liquids 85, no. 3 (May 2000): 269–78. http://dx.doi.org/10.1016/s0167-7322(00)00106-9.

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LAMB, Heather K., Jonathan D. MOORE, Jeremy H. LAKEY, Lisa J. LEVETT, Kerry A. WHEELER, Hugo LAGO, John R. COGGINS, and Alastair R. HAWKINS. "Comparative analysis of the QUTR transcription repressor protein and the three C-terminal domains of the pentafunctional AROM enzyme." Biochemical Journal 313, no. 3 (February 1, 1996): 941–50. http://dx.doi.org/10.1042/bj3130941.

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The AROM protein is a pentadomain protein catalysing steps two to six in the prechorismate section of the shikimate pathway in microbial eukaryotes. On the basis of amino acid sequence alignments and the properties of mutants unable to utilize quinic acid as a carbon source, the AROM protein has been proposed to be homologous throughout its length with the proteins regulating transcription of the genes necessary for quinate catabolism. The QUTR transcription repressor protein has been proposed to be homologous with the three C-terminal domains of the AROM protein and one-fifth of the penultimate N-terminal domain. We report here the results of experiments designed to overproduce the QUTR and AROM proteins and their constituent domains in Escherichia coli, the purpose being to facilitate domain purification and (in the case of AROM), complementation of E. coli aro- mutations in order to probe the degree to which individual domains are stable and functional. The 3-dehydroquinate dehydratase domain of the AROM protein and the 3-dehydroquinate dehydratase-like domain of the QUTR protein were purified in bulk and subjected to comparative CD spectroscopy and fluorescence emission spectroscopy. The CD spectra were found to be virtually superimposable. The fluorescence emission spectra of both domains had the signal from the tryptophan residues almost completely quenched, giving a tyrosine-dominated spectrum for both the AROM- and QUTR-derived domains. This unexpected observation was demonstrated to be due to a highly unusual environment provided by the tertiary structure, as addition of the denaturant guanidine hydrochloride gave a typical tryptophan-dominated spectrum for both domains. The spectroscopy experiments had the potential to refute the biologically-based proposal for a common origin for the AROM and QUTR proteins; however, the combined biophysical data are consistent with the hypothesis. We have previously reported that the AROM dehydroquinate synthase and 3-dehydroquinate dehydratase are stable and functional as individual domains, but that the 5-enol-pyruvylshikimate-3-phosphate synthase is only active as part of the complete AROM protein or as a bi-domain fragment with dehydroquinate synthase. Here we report that the aromA gene (encoding the AROM protein) of Aspergillus nidulans contains a 53 nt intron in the extreme C-terminus of the shikimate dehydrogenase domain. This finding accounts for the previously reported observation that the AROM protein was unable to complement aroE- (lacking shikimate dehydrogenase) mutations in E. coli. When the intron is removed the correctly translated AROM protein is able to complement the E. coli aroE- mutation. An AROM-derived shikimate dehydrogenase domain is, however, non-functional, but function is restored in a bi-domain protein with 3-dehydroquinate dehydratase. This interaction is not entirely specific, as substitution of the 3-dehydroquinate dehydratase domain with the glutathione S-transferase protein partially restores enzyme activity. Similarly an AROM-derived shikimate kinase domain is non-functional, but is functional as part of the complete AROM protein, or as a bi-domain protein with 3-dehydroquinate dehydratase.

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"A structure—function study of dihydrofolate reductase by protein engineering." Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences 317, no. 1540 (April 30, 1986): 405–13. http://dx.doi.org/10.1098/rsta.1986.0050.

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Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10 -1 nm = 10 -10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.

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Quy, Nguyen Thi, Dao Trong Khoa, Duong Thu Huong, Le Thi Thu Hong, and Truong Nam Hai. "Purification of recombinant human interleukin-3 expressed as inclusion bodies in Escherichi coli." Academia Journal of Biology 43, no. 1 (March 31, 2021). http://dx.doi.org/10.15625/2615-9023/13973.

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Human interleukin-3 (IL-3) is a hematopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hematopoietic cells. However, recombinant IL-3 is usually expressed as insoluble form (inclusion bodies) in Escherichia coli cells. This state of protein often shows no bioactivity. Herein, we report a simple method for solubilization, refolding and purification of recombinant human IL-3 expressed in E. coli cells. First, IL-3 was expressed in E. coli JM109 (DE3) after being induced with 0.05 mM IPTG at 25 oC. Under these conditions, IL-3 was produced as inclusion bodies with molecular weight of approximately 15 kDa on SDS-PAGE gel (14%). Next, IL-3 pellet was separated from the host soluble proteins using sonication followed centrifugation. Then, two strong denaturants such as urea or guanidine hydrochloride were used to test solubilization of the insoluble IL-3. After that, the resulting soluble IL-3 was renatured and subjected to gel filtration chromatography to collect purified IL-3 protein. Our results showed that fractionates contained a single band of IL-3 with recovery rate of about 30%. Several characteristics of recombinant IL-3 were then analyzed. The cytokine IL-3 showed its high purity with a sharp peak on RP-HPLC chromatagram. The Western blot showed a clear signal band on PVDF membrane to demonstrate its right antigenecity against human IL-3 antibody. Besides, amino acid sequence of this cytokine was confirmed by mass spectrophotometry method. The purified IL-3 cytokine is a potential material for further tests.

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Wang, Bai, Wei Tang, Peng Zhang, and Qun Wei. "Identification and characterization of the core region of protein phosphatase-1." Biologia 67, no. 2 (January 1, 2012). http://dx.doi.org/10.2478/s11756-012-0009-x.

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AbstractPP1, PP2A and PP2B all belong to PPP family of serine/threonine protein phosphatases. Their primary structures are highly conserved, particularly in the catalytic domain. In order to obtain correlative information about this conserved region, we constructed N-, C-deletion and N/C double-deletion mutants. We found that the N- and Csingle-deletion mutants exhibited higher enzymatic activities, while specific activity of N/C double-deletion mutant PP1 (9-306) did not notably change. The results of kinetics analysis showed that kcat and kcat/Km increased about 16-fold in the single-deletion mutants; while the two parameters of the double-deletion were lower than the single-deletions. We further explored stability of all mutants in existing denaturant guanidine hydrochloride (GdnHCl). It was noticeable that stability of PP1-(9-306) in all mutants was the highest. We speculated that PP1-(9-306) maybe retains a compact spherical structure, thus accordingly affected molecular catalysis. On the other hand the structures of single-deletion mutants were relatively relaxed, which were able to bind substrate easily, so activities of single-deletion mutants were higher than that of double-deletion mutant. We therefore deduced that PP1-(9-306) may be close to core region of PP1 molecule. In order to further solidify this idea, we used fluorescence spectra method to explore changes of space conformation. We found that emission peaks of all single-deletions were blue shifted in different degree in the absence of denaturant, while emission peak of N/C double-deletion mutant did not change obviously compared with that of the wild-type PP1. Conformation change of N/C double-deletion mutant was significantly less than those of single-deletion mutants in different GdnHCl concentration.

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Kang, Sang-Gyun, Zhuang Zhuang Han, Nathalie Daude, Emily McNamara, Serene Wohlgemuth, Laura Molina-Porcel, Jiri G. Safar, Sue-Ann Mok, and David Westaway. "Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope." BMC Biology 19, no. 1 (September 9, 2021). http://dx.doi.org/10.1186/s12915-021-01132-y.

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Abstract Background The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. Results As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. Conclusions Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

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Journal articles on the topic 'Guanidine hydrochloride; Denaturants'

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