Heat capacity and conformation of proteins in the denatured state

Privalov, Peter L.; Tiktopulo, E. I.; Venyaminov, Sergei Yu.; Griko, Yu.V.; Makhatadze, George I.; Хечинашвили, Н. Н.

doi:10.1016/0022-2836(89)90318-5

Cited by 320 publications

(211 citation statements)

References 42 publications

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“…For instance, fully reduced bovine pancreatic trypsin inhibitor (BPTI) has a far-UV CD spectrum characteristic of a random coil (Kosen et al, 1981). The same was reported for bovine ribonuclease (Galat et al, 1981) or lysozyme (Privalov et al, 1989;Radford et al, 1991) with their disulfide bonds disrupted. In these experiments, however, the cysteine residues of the unfolded reduced proteins had to be carboxymethylated to avoid their reoxidation during studies at equilibrium.…”

Section: Discussionmentioning

confidence: 68%

“…Even after optimizing the reoxidation conditions, a renaturation half time of several minutes was found for reduced HEWL (Wetlaufer et al, 1974) as compared to a few seconds only for the nonreduced protein (Kuwajima et al, 1985). Moreover, reduction of disulfide bonds in HEWL (Privalov et al, 1989; Radford et al, 1991), as in other proteins (Creighton, 1975(Creighton, , 1988; Kuroki et al, 1992), is known to decrease the stability of the native protein. The results described in the present report might therefore appear, at first sight, as trivial.…”

Section: Discussionmentioning

confidence: 99%

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Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme

Goldberg

Guillou

1994

Protein Science

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To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the final conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure. Keywords: CD; disulfide bonds; folding intermediates; lysozymeIn a recent study on the refolding of hen egg white lysozyme (HEWL) we observed that an intermediate with a far-UV circular dichroism spectrum compatible with native-like secondary structure was formed in less than 4 ms (Chaffotte et al., 1992). At this early stage of the folding process, the polypeptide chain has no defined tertiary structure as judged from the lack of near-UV CD in the aromatic and disulfide bond regions. Moreover, pulsed proton exchange followed by NMR identification of the protected protons showed that, at this stage, no proton involved in tertiary contacts and a very small fraction only of the backbone amide protons were protected against exchange with the solvent (Radford et al., 1992). Thus, a "molten globule" with native-like secondary structure is formed within the first 4 ms of folding of HEWL. Are disulfide bonds involved in this rapid folding step? We consider 2 alternative models for the effect of correct disulfide bonds on the formation of early folding intermediates of hen lysozyme, when its disulfide bonds are left intact during unfolding:Model I : Early intermediates do not involve correct tertiary contacts. The stability, and also the rate of formation of these intermediates, is the same whether or not correct disulfide bonds are present.Model 2: Although the probes used thus far to characterize early intermediates in a variety of proteins have detected only secondary structure at such early stages of the folding process,

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Section: Discussionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme

Goldberg

Guillou

1994

Protein Science

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“…These functions do not differ significantly from those for RNase A unfolding (Privalov et al, 1989), but they differ significantly from those of myoglobin unfolding, which has a much larger heat capacity increment than barnase or RNase A.…”

Section: The Entropy and Gibbs Energy Of Denaturationmentioning

confidence: 73%

“…The partial specific heat capacity of the native barnase is 1.49 J K" g-I at 25 "C, which is typical for compact globular proteins at this temperature (Privalov et al, 1989). With increasing temperature it increases linearly with a slope of about 0.10 kJ K-2 mol-'.…”

Section: The Partial Heat Capacitymentioning

confidence: 87%

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 E values in entries 48-57 are from Kalnin et al (1990). Entries 58-66 are from the Privalov lab (Privalov et al, 1989;Griko et al, 1994;Yu et al, 1994). For barnase and mutants, entry 67 is from Lees and Hartley (1966), and 68-72 are from Loewenthal et al (1991).…”

Section: Cn Pace Et Almentioning

confidence: 99%

How to measure and predict the molar absorption coefficient of a protein

et al. 1995

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The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319-3261 and is based on data from Edelhoch [ , Biochemistry 6:1948.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, These ~(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict E .

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Heat capacity and conformation of proteins in the denatured state

Cited by 320 publications

References 42 publications

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme

Thermodynamics of barnase unfolding

How to measure and predict the molar absorption coefficient of a protein

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