Urea denaturation of barnase: pH dependence and characterization of the unfolded state

Cn, Pace; Laurents, Douglas V.; Re, Erickson

doi:10.1021/bi00125a013

Cited by 139 publications

(137 citation statements)

References 39 publications

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“…The results suggest that between pH 4 and 2, 3.5 protons are bound on average. This value is close to that obtained previously by Hartley (1969) and by Pace et al (1992).…”

Section: Influence Of P H On the Thermostabilitysupporting

confidence: 93%

“…As follows from the pH dependence of the transition temperature, r, (Fig. 2), barnase is most stable in the region of pH in which it shows a maximal activity of nucleotide hydrolysis (4.5-6.5) (Hartley, 1969;Mossakowska et al, 1989;Pace et al, 1992). In this pH region thermostability of barnase does not depend noticeably on pH, which means that here barnase does not lose or gain protons upon unfolding.…”

Section: Influence Of P H On the Thermostabilitymentioning

confidence: 81%

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Thermodynamics of barnase unfolding

et al. 1994

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The thermodynamics of barnase denaturation has been studied calorimetrically over a broad range of temperature and pH. It is shown that in acidic solutions the heat denaturation of barnase is well approximated by a 2-state transition. The heat denaturation of barnase proceeds with a significant increase of heat capacity, which determines the temperature dependencies of the enthalpy and entropy of its denaturation. The partial specific heat capacity of denatured barnase is very close to that expected for the completely unfolded protein. The specific denaturation enthalpy value extrapolated to 130 "C is also close to the value expected for the full unfolding. Therefore, the calorimetrically determined thermodynamic characteristics of barnase denaturation can be considered as characteristics of its complete unfolding and can be correlated with structural features -the number of hydrogen bonds, extent of van der Waals contacts, and the surface areas of polar and nonpolar groups. Using this information and thermodynamic information on transfer of protein groups into water, the contribution of various factors to the stabilization of the native structure of barnase has been estimated. The main contributors to the stabilization of the native state of barnase appear to be intramolecular hydrogen bonds. The contributions of van der Waals interactions between nonpolar groups and those of hydration effects of these groups are not as large if considered separately, but the combination of these 2 factors, known as hydrophobic interactions, is of the same order of magnitude as the contribution of hydrogen bonding.Keywords: barnase; hydrogen bonding; hydrophobic interactions; scanning microcalorimetry Barnase attracts the special interest of researchers engaged in studying protein folding because it is one of the smallest globular proteins (1 10 amino acid residues; see Kinemage 1) that does not have disulfide crosslinks but is quite stable and unfolds reversibly. The latter circumstance is very important for quantitative analysis of this process by equilibrium thermodynamics, specifying the stability of the native state in energetic terms.In solutions close to neutral pH, reversible unfolding of barnase by denaturants and temperature appears to be a 2-state transition (Hartley, 1968(Hartley, , 1975Makarov et al., 1993). Our earlier unpublished calorimetric studies of the heat denaturation of barnase, carried out at the Protein Research Institute in Russia in 1989, showed also that this process is approximated well by a 2-state transition. However, later in 1993 the paper of Makarov et al. (1993) ing subdomains. Analysis of the 3-dimensional structure of these proteins does indeed reveal 2 hydrophobic clusters (see Kinemage 1;Serrano et al., 1992). Therefore, the mode of barnase denaturation required detailed investigation. Because there was also some deviation between our estimates of the enthalpy of denaturation and that of Makarov et al. (1993), we felt it necessary to repeat calorimetric measurements on barnase to ...

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“…The results suggest that between pH 4 and 2, 3.5 protons are bound on average. This value is close to that obtained previously by Hartley (1969) and by Pace et al (1992).…”

Section: Influence Of P H On the Thermostabilitysupporting

confidence: 93%

Section: Influence Of P H On the Thermostabilitymentioning

confidence: 81%

Thermodynamics of barnase unfolding

et al. 1994

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“…This is an important observation since it shows that DSE electrostatic interactions can make large contributions to protein stability. Significant DSE electrostatic interactions have also been observed in RNase H and ribonuclease Sa [29][30][31]. The case of RNase H is particularly interesting because it provides an example of the possible functional consequences of DSE structure.…”

Section: Electrostatic Interactions Are Found In the Denatured State mentioning

confidence: 99%

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Cho¹,

Sato²,

Horng³

et al. 2008

Archives of Biochemistry and Biophysics

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It is now recognized that the denatured state ensemble (DSE) of proteins can contain significant amounts of structure, particularly under native conditions. Well-studied examples include small units of hydrogen bonded secondary structure, particularly helices or turns as well hydrophobic clusters. Other types of interactions are less well characterized and it has often been assumed that electrostatic interactions play at most a minor role in the DSE. However, recent studies have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE. These can include surprisingly specific non-native interactions that can even persist in the transition state for protein folding. DSE electrostatic interactions can be energetically significant and their modulation either by mutation or by varying solution conditions can have a major impact upon protein stability. pH dependent stability studies have shown that electrostatic interactions can contribute up to 4 kcal mol −1 to the stability of the DSE.

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“…a Swint and Robertson, 1993;' O'Neil et al, 1995;Hurle et al, 1990;Viguera et al, 1994; e Jackson et al, 1993;'Akke and Forsen, 1990;Khorasanizadeh et al, 1993;Wintrode et al, 1994;Scholtz, 1995;J Agashe and Udgaonkar, 1995;Lim et al, 1992; ' Marqusee and Sauer, 1994;McLendon and Smith, 1978;Hagihara et al, 1994; O Privalov and Gill, 1988;P Pace et al, 1990;Shirley et al, 1992;Yu et al, 1994; ' Bowie and Sauer, 1989; ' Egan et al, 1993;Ramdas et al, 1986;"Bryant et al, 1985; " Cohen and Pielak, 1994;x Kelley et al, 1987;Santor0 and Bolen, 1992;'Clarke and Fersht, 1993; aa Pace et al, 1992;" Griko et al, 1994; cc Greene and Pace, 1974;dd Munson et al, 1994;Filimonov et al, 1993; f f Saito and Wada, 1983; gg Ahmad and Bigelow, 1982; hh Taniyama et al, 1992; I' Herning et al, 1992;'' Ropson et al, 1990; kk Shortle and Meeker, 1986;"Carra et al, 1994; mm Craig et al, 1987; "" Makhatadze et al, 1994;O0 De Young et al, 1993...…”

Section: Aasamentioning

confidence: 99%

“…a Data on Trp and Tyr accessibility are from Pace et al (1992). Effects on AASA for folding are calculated as described in the text.…”

Section: Correlations Of Rn Values and Heat Capacity Changes With Aasamentioning

confidence: 99%

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

1995

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Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (AC,), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two-state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and AC,, Keywords: denaturation; guanidine hydrochloride; heat capacity changes; m values; protein folding; protein stability; solvent-accessible surface area; urea It has been known for many years that proteins can be unfolded in aqueous solution by high concentrations of certain reagents such as guanidine hydrochloride or urea. Denaturation with these chemicals is one of the primary ways of measuring the conformational stability of proteins and comparing the stabilities of mutant proteins. The use of these two denaturants is extremely widespread (Pace, 1986), even though the exact nature of the molecular interaction of denaturant molecules with protein surfaces is not well understood. It is known from solubility and transfer experiments with model compounds that the interaction of urea and Gdn HCI with the constituent groups of proteins is more favorable than the interaction of those groups with water (Tanford, 1970). These denaturants alter the equi-~"

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Urea denaturation of barnase: pH dependence and characterization of the unfolded state

Cited by 139 publications

References 39 publications

Thermodynamics of barnase unfolding

Thermodynamics of barnase unfolding

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

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