Progress in the Development of an Alternative Approach to Macromolecular Crystallization

Larson, Steven B.; Day, John; Nguyen, C.; Cudney, Robert; McPherson, Alexander

doi:10.1021/cg800174n

Cited by 22 publications

(23 citation statements)

References 26 publications

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“…As in previous investigations (Larson et al, 2007(Larson et al, , 2008 smallmolecule components of the crystallization 'cocktails' were found as integral components of the crystals and again were involved in macromolecular interfaces in the lattice. Thus, they provided additional lattice contacts and possibly stabilizing interactions.…”

Section: Discussionsupporting

confidence: 67%

“…The crystal screening experiments that yielded the complex crystals have been described in detail elsewhere (Larson et al, 2008). The crystals were grown by vapor diffusion at room temperature in 96-well Intelli-Plates (Hampton Research, Aliso Viejo, California, USA) with 90 ml reservoirs of 25% PEG 3350 in water.…”

Section: Methodsmentioning

confidence: 99%

“…We have reported in previous papers (McPherson & Cudney, 2006;Larson et al, 2007Larson et al, , 2008 on the promotion of the crystallization of macromolecules by mixtures, or 'cocktails', of a variety of conventional small molecules. In nine cases, we were able to show (Larson et al, 2007(Larson et al, , 2008) that these small molecules mediated interactions between macromolecules in the crystals, provided lattice interactions and were visible in difference electron-density maps. In all of these cases, we found the small molecules to be well ordered in the crystals and held firmly in place by hydrogen bonds and electrostatic interactions.…”

Section: Introductionmentioning

confidence: 99%

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High-resolution structure of proteinase K cocrystallized with digalacturonic acid

Larson

Day

Nguyen³

et al. 2009

Acta Cryst Sect F

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PDB Reference: proteinase K-digalacturonic acid complex, 3dyb, r3dybsf.Proteinase K, a subtilisin-like fungal protease, was crystallized from a cocktail of small molecules containing digalacturonic acid (DGA). The crystal structure was determined to 1.32 Å resolution and refined to an R factor of 0.158. The final model contained, beside the protein, two calcium ions, 379 water molecules, a molecule of DGA and a partially occupied HEPES molecule. The DGA molecule has one sugar moiety disposed exactly on a crystallographic twofold axis; the second ring was not observed. The DGA molecule is bound to two protein molecules across the twofold axis through hydrogen-bonding networks involving Ser150 and water molecules. One of the calcium-ion sites has not been reported previously. This study further illustrates the involvement of small molecules in the crystallization of macromolecules through their ability to form intermolecular lattice interactions.

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Section: Discussionsupporting

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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High-resolution structure of proteinase K cocrystallized with digalacturonic acid

Larson

Day

Nguyen³

et al. 2009

Acta Cryst Sect F

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“…Numerous cases have, however, been reported in which small, and sometimes large, molecules were observed to make crucial interactions between macromolecules in the crystal that either helped to guide or secure formation of the lattice (Larson et al, 2008;McPherson & Cudney, 2006). Such small molecules sometimes had a physiological basis for their unexpected presence, but frequently did not.…”

Section: Factors Affecting Crystallization and The Inclusion Of Additmentioning

confidence: 99%

Introduction to protein crystallization

McPherson

Gavira

2013

Acta Crystallogr F Struct Biol Commun

358

323

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Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

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“…Small molecules have been recognized as often being crucial for protein crystallization, but they have seldom been identified within the crystal structure. 67,68 There are also experimental results showing that long alkyl chain imidazolium-based IL ([C 14 MIM]Br) in fact binds to the surface of a globular protein bovine serum aluminum (BSA). 69 In agreement with the mechanisms of IL-protein interactions discussed in our work, it has been found that, at lower concentration, [C 14 MIM]Br binds to the BSA surface due to electrostatic attraction while at higher IL concentration the mechanism is that of hydrophobic interaction.…”

Section: Ils In the Task Of Crystallization: Reaction Environment Vsmentioning

confidence: 99%

Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions

et al. 2012

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We have performed experiments on the crystallization of two low molecular weight, positively charged proteins, lysozyme and ribonuclease A, using ionic liquids as either crystallization additives or, in particular cases, as precipitating agents. The ionic liquids (ILs) have been ordered according to their salting-in/out ability and the relative position of these ionic liquids in this ranking has been rationalized by considering their hydration properties (positive-negative, hydrophobichydrophilic). The ability to screen the effective charge of cationic proteins and aid protein nucleation (salting-out) has been shown to be superior for large polarizable anions with low charge density, negatively hydrated-Cl 2 , Br 2 , [SCN] 2 , methane-[C 1 SO 3 ] 2 and ethanesulfonates [C 2 SO 3 ] 2 , than for anions with a relatively stable hydration shell, positively hydrated-lactate [Lac] 2 , butylsulfonate [C 4 SO 3 ] 2 and acetate [Ac] 2. Upon increasing the background salt concentration, where electrostatic interactions are already effectively screened, the ability of the IL ions to stabilize proteins in solution (salting-in) has been shown to increase as the ions are likely to migrate to the non-polar protein surface and lower protein-water interfacial tension. This tendency is enhanced as the focus moves from those ions with positively hydrated hydrophilic compartments (e.g. [Ac] 2) to those with negatively hydrated groups (e.g. [C 1 SO 3 ] 2) and the prevailing hydrophobic hydration (e.g. [C 4 SO 3 ] 2). The observed inversion in the relative effect of ILs on protein crystallization with increasing ionic strength of the aqueous media has been interpreted as the differing effects of ion adsorption: charge screening and interfacial tension modification. Moreover, this work can further help in our understanding of the influence of ionic liquids on conformational changes of biomacromolecules in solution. Identification of the specific incorporation sites for choline and acetate ions, localized in two lysozyme crystals grown in pure IL solutions without any buffer or inorganic precipitant, can give us some insight into the role of the ionic liquid ions in protein structure development.

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Progress in the Development of an Alternative Approach to Macromolecular Crystallization

Cited by 22 publications

References 26 publications

High-resolution structure of proteinase K cocrystallized with digalacturonic acid

High-resolution structure of proteinase K cocrystallized with digalacturonic acid

Introduction to protein crystallization

Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions

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