A structural basis for the interaction of urea with lysozyme

Pike, Ashley C.W.; Acharya, K. Ravi

doi:10.1002/pro.5560030419

Cited by 78 publications

(65 citation statements)

References 20 publications

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“…Unlike earlier studies, no electron density corresponding to denaturant molecules were visible in any of the urea soaked structures. Also the decrease in B-factors observed earlier, for RNase A, 22 DHFR, 22 and lysozyme 21 was not observed in the present work with the exception of some regions in the 1.5 M** structure. This is probably because the present studies use denaturant concentrations close to the corresponding Cm in solution while the earlier studies were all carried out at denaturant concentrations well below the Cm.…”

Section: Discussionmentioning

confidence: 39%

“…Several lysozyme structures were solved at high resolution (1.4-1.6 Å ) and in the presence of high concentrations (up to 9 M) of urea. 21 However the crystal structures do not give any structural insight into lysozyme unfolding as the addition of urea actually increases the order in the crystal structure by stabilizing loop regions and decreasing the overall B-factors. The protein structure remains unchanged in the presence of urea.…”

Section: Introductionmentioning

confidence: 99%

“…X-ray crystallography has also been used to structurally characterize proteins 21,22 and peptide models 23,24 in the presence of denaturants. Protein crystals grown in the absence of denaturants can be soaked in solutions containing denaturants.…”

Section: Introductionmentioning

confidence: 99%

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X‐ray crystallographic studies of the denaturation of ribonuclease S

Ratnaparkhi

Varadarajan

1999

Proteins

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In an attempt to view the onset of urea denaturation in ribonuclease we have collected X-ray diffraction data on ribonuclease S crystals soaked in 0, 1.5, 2, 3, and 5 molar urea. At concentrations above 2 M urea, crystals were stabilized by glutaraldehyde crosslinking. We have also collected data on ribonuclease S crystals at low pH in an attempt to study the onset of pH denaturation. The resolution of the datasets range from 1.9 to 3.0 Å . Analysis of the structures reveals an increase in disorder with increasing urea concentration. In the 5 M urea structure, this increase in disorder is apparent all over the structure but is larger in loop and helical regions than in the ␤ strands. The low pH structure shows a very similar pattern of increased disorder. In addition there is a major change in the position of the main chain (G 1 Å ) in the 65-72 turn region. This region has previously been shown to be involved in one of the initial steps of unfolding in the reduction of ribonuclease A. Crystallographic analyses in the presence of denaturant, when combined with controlled crosslinking, can thus provide detailed structural information that is related to the initial steps of unfolding in solution. Proteins 1999;36:282-294.

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Section: Discussionmentioning

confidence: 39%

Section: Introductionmentioning

confidence: 99%

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X‐ray crystallographic studies of the denaturation of ribonuclease S

Ratnaparkhi

Varadarajan

1999

Proteins

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“…In the inset the k diss -GdnHCl gradient is shown to connect the slope of the stoppedflow unfolding rate of the protein, k u , as a function of GdnHCl measured at 10°C. k u values were measured by changes in tryptophan fluorescence (]) and 430 nm Soret heme absorbance(3). The arrow points to the C m of equilibrium unfolding of ferrocytochrome c at 10°C, 0.1 M phosphate, pH 7.…”

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confidence: 99%

Protein Stabilization by Urea and Guanidine Hydrochloride

2002

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The urea, guanidine hydrochloride, salt, and temperature dependence of the rate of dissociation of CO from a nonequilibrium state of CO-bound native ferrocytochrome c has been studied at pH 7. The heme iron of ferrocytochrome c in the presence of denaturing concentrations of guanidine hydrochloride (GdnHCl) and urea prepared in 0.1 M phosphate, pH 7, binds CO. When the unfolded protein solution is diluted 101-fold into CO-free folding buffer, the protein chain refolds completely, leaving the CO molecule bonded to the heme iron. Subsequently, slow thermal dissociation of the CO molecule yields to the heme coordination of the native M80 ligand. Thus, the reaction monitors the rate of thermal conversion of the CO-liganded native ferrocytochrome c to the M80-liganded native protein. The rate of this reaction, k(diss), shows a characteristic dependence on the presence of nondenaturing concentrations of the denaturants in the reaction medium. The rate decreases by approximately 1.9-3-fold as the concentration of GdnHCl in the refolding medium increases from nearly 0 to approximately 2.1 M. Similarly, the rate decreases by 1.8-fold as the urea concentration is raised from 0.l to approximately 5 M. At still higher concentrations of the denaturants the denaturing effect sets in, the protein is destabilized, and hence the CO dissociation rate increases sharply. The activation energy of the reaction, E(a), increases when the denaturant concentration in the reaction medium is raised: from 24.1 to 28.3 kcal mol(-1) for a 0.05-2.1 M rise in GdnHCl and from 25.2 to 26.9 kcal mol(-1) for a 0.1-26.9 M increase in urea. Corresponding to these increases in denaturant concentrations are also increases in the activation entropy, S(diss)/R, where R is the gas constant of the reaction. The denaturant dependence of these kinetic and thermodynamic parameters of the CO dissociation reaction suggests that binding interactions with GdnHCl and urea can increase the structural and energetic stability of ferrocytochrome c up to the limit of the subdenaturing concentrations of the additives. NaCl and Na(2)SO(4), which stabilize proteins through their salting-in effect, also decrease the rate with a corresponding increase in activation entropy of CO dissociation from CO-bound native ferrocytochrome c, lending support to the view that low concentrations of GdnHCl and urea stabilize proteins. These results have direct relevance to the understanding and interpretation of the free energy-denaturant relationship and protein folding chevrons.

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“…Early crystallographic work suggested that urea stabilizes the flexible parts of folded proteins, and relaxes the crystal packing contacts. 10 Bhuyan 11 has also shown, using different methods, that low (mM) concentrations of urea may stabilize proteins. The thermal dissociation of CO from ferrocytochrome c was slowed at low 'salting-in' concentrations of urea or guanidine hydrochloride.…”

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confidence: 97%

Urea as a protein destabilizing agent in electrospray ionisation

Donald

Collado

Galka

et al. 2009

Rapid Comm Mass Spectrometry

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Urea is well known as a denaturant of proteins, but there is also evidence that millimolar amounts of urea may in fact stabilize protein complexes. Advances in mass spectrometric analysis have given us the opportunity to test the effect of urea on several noncovalent complexes in buffered solutions. We expected to see lower charge states if folded proteins were more compact (and therefore more stable), and higher charge states if the proteins were denatured. We have found that mM urea interferes with some noncovalent interactions, and that the extent of interference depends on the specific protein complex. The difference seems to be related to the type of interactions, with weak ones, such as H-bonds, more sensitive to urea. Examples show that a quick check with urea may give some insights into protein stability in the mass spectrometer.

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A structural basis for the interaction of urea with lysozyme

Cited by 78 publications

References 20 publications

X‐ray crystallographic studies of the denaturation of ribonuclease S

X‐ray crystallographic studies of the denaturation of ribonuclease S

Protein Stabilization by Urea and Guanidine Hydrochloride

Urea as a protein destabilizing agent in electrospray ionisation

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