Studies on bovine pancreatic ribonuclease A and model compounds in aqueous 2-methyl-2,4-pentanediol

Pittz, Eugene P.; Bello, Jake

doi:10.1016/0003-9861(71)90156-1

Cited by 36 publications

(19 citation statements)

References 48 publications

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“…The concentrations, rather than the activities, of the amino acids are used because activities for three-component systems are very difficult or impossible to obtain with any accuracy (13)(14)(15). Following the lead of others, we report the ⌬G tr values as apparent transfer Gibbs energy changes (12,(16)(17)(18)(19)(20)(21). Table 1 presents solubility limits and transfer Gibbs energy changes of all amino acids except cysteine and cystine from water to 1 M proline solution.…”

Section: Resultsmentioning

confidence: 99%

Osmolyte-driven contraction of a random coil protein

Bolen

1998

Proc. Natl. Acad. Sci. U.S.A.

288

261

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The Stokes radius characteristics of reduced and carboxamidated ribonuclease A (RCAM RNase) were determined for transfer of this ''random coil'' protein from water to 1 M concentrations of the naturally occurring protecting osmolytes trimethylamine N-oxide, sarcosine, sucrose, and proline and the nonprotecting osmolyte urea. The denatured ensemble of RCAM RNase expands in urea and contracts in protecting osmolytes to extents proportional to the transfer Gibbs energy of the protein from water to osmolyte. This proportionality suggests that the sum of the transfer Gibbs energies of individual parts of the protein is responsible for the dimensional changes in the denatured ensemble. The dominant term in the transfer Gibbs energy of RCAM RNase from water to protecting osmolytes is the unfavorable interaction of the osmolyte with the peptide backbone, whereas the favorable interaction of urea with the backbone dominates in RCAM RNase transfer to urea. The side chains collectively favor transfer to the osmolytes, with some protecting osmolytes solubilizing hydrophobic side chains as well as urea does, a result suggesting there is nothing special about the ability of urea to solubilize hydrophobic groups. Protecting osmolytes stabilize proteins by raising the chemical potential of the denatured ensemble, and the uniform thermodynamic force acting on the peptide backbone causes the collateral effect of contracting the denatured ensemble. The contraction decreases the conformational entropy of the denatured state while increasing the density of hydrophobic groups, two effects that also contribute to the ability of protecting osmolytes to force proteins to fold.The adaptation of certain higher organisms to harsh environments is enabled by the intracellular presence of small organic solutes (osmolytes) that protect proteins and other cell components from the denaturing environmental stresses (1). Arakawa and Timasheff (2) have shown that the osmolytes act by raising the chemical potential of the denatured state relative to the native state, thereby increasing the (positive) Gibbs energy difference (⌬G) between the native and denatured ensembles. A pictorial description of the results of Arakawa and Timasheff is presented in the Gibbs energy diagram below, where ⌬G 1 is the unfolding Gibbs energy difference between native (N aq ) and unfolded (U aq ) protein in aqueous buffer solution, and ⌬G 3 is the Gibbs energy change for the same reaction in the presence of osmolytes. Transfer of N aq or U aq from water to osmolyte solution (N os and U os respectively) raises the chemical potential of the unfolded ensemble (⌬G 2 ) much more than it does that of the native state ensemble (⌬G 4 ), resulting in a greater stability of the protein in the osmolyte solution than in buffer, i.e., ⌬G 3 Ͼ⌬G 1 . We have determined the transfer Gibbs energy changes of amino acid side chains and peptide backbone from water to solutions of the osmolytes trimethylamine N-oxide (TMAO), sarcosine, and sucrose (3, 4). From knowledge of these tran...

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Section: Resultsmentioning

confidence: 99%

Osmolyte-driven contraction of a random coil protein

Bolen

1998

Proc. Natl. Acad. Sci. U.S.A.

288

261

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“…It is tempting to hypothesize that the coalescence of the detergent may be brought about by the precipitant 2-methyl-2, 4-pentanediol, which has been shown to act as an amphiphile (Kita et al, 1994) and which has a marked nonpolar character (Pittz and Bello, 1971). Moderate amounts of organic amphiphiles have been shown to be advantageous for membrane protein crystallisation, and their beneficial effect was attributed to increased micellar fluidity and deformability and decreased micellar size (Garavito et al, 1986;Marone et al, 1999;Michel, 1982Michel, , 1983Papiz et al, 1989;Timmins et al, 1991).…”

Section: Crystal Packing and Crystal Growthmentioning

confidence: 99%

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Snijder¹,

Timmins²,

Kalk³

et al. 2003

Journal of Structural Biology

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The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H 2 O=D 2 O contrast. From the neutron diffraction at five contrasts, the 12 A A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic b-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A A. The enzymeÕs active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzymeÕs dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.

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“…For example, 2-methyl-2,4-pentanediol (MPD) is a highly effective protein crystallizing agent and precipitates proteins in the native state [17,18]. Moderate concentration (10-40%) of ethanol has been used as one of the efficient methods to fractionate plasma proteins into various functional therapeutic products [19][20][21].…”

Section: Introductionmentioning

confidence: 99%