Crystallization of yeast triose phosphate isomerase from polyethylene glycol. Protein crystal formation following phase separation.

Alber, Tom; Hartman, Fred C.; Johnson, RA; Petsko, Gregory A.; Tsernoglou, Demetrius

doi:10.1016/s0021-9258(19)69972-2

Cited by 26 publications

(4 citation statements)

References 39 publications

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“…It is also well known that protein crystallization may involve not only the favourable solid -liquid phase separation but also other metastable phases such as liquid -liquid phase separation (LLPS). LLPS has been shown, both experimentally (Alber et al 1981;Vivares et al 2005;Dumetz et al 2008) and theoretically via the second virial coefficient (Haas & Drenth 1998Vliegenthart & Lekkerkerker 2000), to be an important process in the nucleation and growth of protein crystals. Similarly, the nucleation and assembly pathway of viral capsid subunits may ultimately be revealed through theoretical consideration once the subunit binding interactions have been determined directly from experiments.…”

Section: Discussionmentioning

confidence: 99%

Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres

Chuan

Fan

Lua

et al. 2009

J. R. Soc. Interface.

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Viral self-assembly is of tremendous virological and biomedical importance. Although theoretical and crystallographic considerations suggest that controlled conformational change is a fundamental regulatory mechanism in viral assembly, direct proof that switching alters the thermodynamic attraction of self-assembling components has not been provided. Using the VP1 protein of polyomavirus, we report a new method to quantitatively measure molecular interactions under conditions of rapid protein self-assembly. We show, for the first time, that triggering virus capsid assembly through biologically relevant changes in Ca 2þ concentration, or pH, is associated with a dramatic increase in the strength of protein molecular attraction as quantified by the second virial coefficient (B 22 ). B 22 decreases from 22.3 Â 10 24 mol ml g 22(weak protein -protein attraction) to 22.4 Â 10 23 mol ml g 22 (strong protein attraction) for metastable and Ca 2þ -triggered self-assembling capsomeres, respectively. An assemblydeficient mutant (VP1CD63) is conversely characterized by weak protein -protein repulsion independently of chemical change sufficient to cause VP1 assembly. Concomitant switching of both VP1 assembly and thermodynamic attraction was also achieved by in vitro changes in ammonium sulphate concentration, consistent with protein salting-out behaviour. The methods and findings reported here provide new insight into viral assembly, potentially facilitating the development of new antivirals and vaccines, and will open the way to a more fundamental physico-chemical description of complex protein self-assembly systems.

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Section: Discussionmentioning

confidence: 99%

Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres

Chuan

Fan

Lua

et al. 2009

J. R. Soc. Interface.

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“…The cocrystallization of yeast TIM and PGA has been previously reported (Alber et al, 1981b). Yeast triosephosphate isomerase (Sigma Chemical Co., type I) was exhaustively dialyzed against a solution of 0.2 M Tris (pH 6.8) containing 1 mM EDTA and 1 mM mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-.ANG. resolution: implications for catalysis

Lolis

Petsko²

1990

Biochemistry

247

254

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The binding of the transition-state analogue 2-phosphoglycolate to triosephosphate isomerase from yeast has been investigated crystallographically. An atomic model of the enzyme-inhibitor complex has been refined against data to 2.5-A resolution to a final R factor of 0.18. The interactions between the inhibitor and enzyme have been analyzed. The inhibitor forms hydrogen bonds to the side chains of His 95 and Glu 165. The latter hydrogen bond confirms that Glu 165 is protonated upon PGA binding. The structure of the complexed enzyme has been compared to that of the unbound form of the enzyme, and conformational changes have been observed: the side chain of Glu 165 moves over 2 A and a 10-residue flexible loop moves over 7 A to close over the active site. Spectroscopic results of phosphoglycolic acid binding to triosephosphate isomerase that have been amassed over the years are also explained in structural terms. The implications for catalysis are noted.

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“…Crystallization and Heavy Atom Search. The crystallization of yeast TIM has been reported previously (Alber et al, 1981b). A 20 mg/mL sample of yeast triosephosphate isomerase in 12% saturated ammonium sulfate, 1 mM EDTA, 1 mM mercaptoethanol, and 50 mM Tris buffer, pH 7.5, was crystallized by the addition of poly(ethylene glycol) of average molecular weight 4000 to a final concentration of about 16% by either vapor diffusion or batch methods.…”

Section: Methodsmentioning

confidence: 99%

Structure of yeast triosephosphate isomerase at 1.9-.ANG. resolution

Lolis¹,

Alber²,

Davenport³

et al. 1990

Biochemistry

254

203

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The structure of yeast triosephosphate isomerase (TIM) has been solved at 3.0-A resolution and refined at 1.9-A resolution to an R factor of 21.0%. The final model consists of all non-hydrogen atoms in the polypeptide chain and 119 water molecules, a number of which are found in the interior of the protein. The structure of the active site clearly indicates that the carboxylate of the catalytic base, Glu 165, is involved in a hydrogen-bonding interaction with the hydroxyl of Ser 96. In addition, the interactions of the other active site residues, Lys 12 and His 95, are also discussed. For the first time in any TIM structure, the "flexible loop" has well-defined density; the conformation of the loop in this structure is stabilized by a crystal contact. Analysis of the subunit interface of this dimeric enzyme hints at the source of the specificity of one subunit for another and allows us to estimate an association constant of 10(14)-10(16) M-1 for the two monomers. The analysis also suggests that the interface may be a particularly good target for drug design. The conserved positions (20%) among sequences from 13 sources ranging on the evolutionary scale from Escherichia coli to humans reveal the intense pressure to maintain the active site structure.

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Crystallization of yeast triose phosphate isomerase from polyethylene glycol. Protein crystal formation following phase separation.

Cited by 26 publications

References 39 publications

Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres

Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres

Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-.ANG. resolution: implications for catalysis

Structure of yeast triosephosphate isomerase at 1.9-.ANG. resolution

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Crystallization of yeast triose phosphate isomerase from polyethylene glycol. Protein crystal formation following phase separation.

Cited by 26 publications

References 39 publications

Virus assembly occurs following a pH- or Ca2+-triggered switch in the thermodynamic attraction between structural protein capsomeres

Virus assembly occurs following a pH- or Ca2+-triggered switch in the thermodynamic attraction between structural protein capsomeres

Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-.ANG. resolution: implications for catalysis

Structure of yeast triosephosphate isomerase at 1.9-.ANG. resolution

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Product

Resources

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Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres

Virus assembly occurs following a pH- or Ca²⁺-triggered switch in the thermodynamic attraction between structural protein capsomeres