Protein dynamics and solvation in aqueous and nonaqueous environments

Hartsough, David S.; Merz, Kenneth M.

doi:10.1021/ja00068a009

Cited by 96 publications

(86 citation statements)

References 3 publications

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“…Studies involving molecular dynamics simulations have suggested that proteins in organic solvents should possess a diminished overall exposed surface area relative to their structures in water (21,22). We observed (Fig.…”

Section: Resultsmentioning

confidence: 52%

“…3C). The simulation studies (21,22) also predict that nonpolar residues will be more exposed and polar residues less exposed in organic solvents compared with water. Although some subtilisin residues agree with this prediction, others do not.…”

Section: Resultsmentioning

confidence: 97%

“…3 B and C). These inconsistencies could have arisen because the simulation studies (21,22) were performed on a different protein, bovine pancreatic trypsin inhibitor, and in a different non-hydrogen-bonding solvent, chloroform. An alternative possibility is that the enzyme is kinetically trapped in the same conformation as in water and consequently cannot reach a minimum energy state of the sort predicted by the molecular dynamics simulations.…”

Section: Resultsmentioning

confidence: 99%

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The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile

Schmitke

Stern

Klibanov

1997

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

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The x-ray crystal structure of the serine protease subtilisin Carlsberg in anhydrous dioxane has been determined to 2.6-Å resolution. The enzyme structure is found to be nearly indistinguishable from the structures previously determined in water and acetonitrile. Small changes in the side-chain conformations between the dioxane and water structures are of the same magnitude as those observed between two structures in different aqueous systems. Seven enzyme-bound dioxane molecules have been detected, each potentially forming at least one hydrogen bond with a subtilisin hydrogen-bond donor or bound water. Two of the bound dioxane molecules are in the active-site region, one in the P2 and another bridging the P1 and P3 pockets. The other five dioxane molecules are located on the surface of subtilisin at interprotein crystal contacts. The locations of the bound solvent in the dioxane structure are distinct from those in the structures in acetonitrile and in water.Enzymatic catalysis in organic medium has emerged as an important area of research in biotechnology and bioorganic chemistry (1). The structure of enzymes in such milieu should aid in the understanding and optimization of this catalysis. Several years ago, we succeeded in determining the first such x-ray crystal structure, that of subtilisin Carlsberg in anhydrous acetonitrile (2). It was found that the structure of the lightly cross-linked enzyme crystal in the anhydrous solvent was virtually indistinguishable from that in water (2, 3). This conclusion was corroborated by subsequent crystallographic studies of two other enzymes-␥-chymotrypsin in hexane (4, 5) and elastase in acetonitrile (6).To date (2-6) only one nonaqueous solvent has been examined for each crystalline enzyme. A rationale has been tentatively adopted (7-9) that if the enzyme structure does not change upon transition from water to acetonitrile or hexane, then it also should not change upon transition to another solvent because the difference in physicochemical properties between two organic solvents should be less than between water and an organic solvent. However, a direct validation of this reasoning until now has been missing.In the present study, we have determined the x-ray crystal structure of lightly cross-linked subtilisin in a second unrelated anhydrous solvent, dioxane. This structure has turned out to be essentially the same as those in water (3) and in acetonitrile (2). Interestingly, the locations of the subtilisin-bound dioxane and acetonitrile molecules are distinct. MATERIALS AND METHODSCrystal Preparation. Subtilisin Carlsberg (serine protease from Bacillus licheniformis, EC 3.4.21.14) was purchased from Sigma. Crystals were grown from an aqueous 330 mM cacodylate buffer (pH 5.6) saturated with Na 2 SO 4 , approximately 13% (10). A single crystal (ϳ0.8 ϫ 0.1 ϫ 0.05 mm) was placed in 1 ml of a 10% glutaraldehyde cross-linking solution containing 30 mM cacodylate buffer (pH 7.5) and 10% Na 2 SO 4 . The glutaraldehyde solution was aged for 3 days at room temperature ...

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Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile

Schmitke

Stern

Klibanov

1997

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

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“…However, this observation cannot be generalized, because for BPTI they found that the deviation was larger for the water simulation. [11,20] In the case of CRL, simulation in CCl 4 leads to a higher number of internal hydrogen bonds (data not shown), indicating that CRL becomes more packed. Increase of internal hydrogen bonds is also related to a decrease of the radius of gyration and solvent-accessible surface area.…”

Section: Structurementioning

confidence: 93%

“…[24] This higher flexibility also may be caused by reduced electrostatic interaction due to dielectric screening [25] upon solvating the polar and charged residues. [26] Lower flexibility of protein was also observed in chloroform, [11,21] hexane, [20,27] methyl hexanoate, [28] dimethylformamide, acetone, butanone, tetrahydrofuran, and cyclohexane.…”

Section: Structurementioning

confidence: 93%

Structure and dynamics of Candida rugosa lipase: the role of organic solvent

2004

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The effect of organic solvent to structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (C α rms deviation 1-1.3 Å). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as rigid body about its average position. While in water the deviations were about 1.6 Å, organic solvent reduced flexibility to 0.9 Å. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus organic solvents stabilize the lid but render the side chains in the hydrophobic substrate binding site more mobile.

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