Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data

Price, W. Nicholson; Chen, Yang; Handelman, Samuel K.; Neely, H.; Manor, Philip C.; Karlin, Richard; Nair, Rajesh; Liu, Jinfeng; Baran, M.C.; Everett, J.K.; Tong, Saichiu N; Forouhar, F.; Swaminathan, Swarup S.; Acton, Thomas; Xiao, Rong; Luft, Joseph R.; Lauricella, Angela; DeTitta, George T.; Rost, Burkhard; Montelione, Gaetano T.; Hunt, J.F.

doi:10.1038/nbt.1514

Cited by 132 publications

(178 citation statements)

References 36 publications

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“…Previous studies have attributed the infrequent participation of Lys in interfaces to the entropic cost of restricting its highly mobile side chain. 14,28 This proposal seems to be incompatible with our results (for the specific but very well-defined The results of this study also emphasize the significant role of surface charges in mediating interactions between proteins, 29,30 and suggest that charges, as a constraint in determining proteinprotein interfaces, might be modulated (or reduced) to control biomolecular recognition. We showed that acetylation of positively charged lysine residues gives rise to new protein-protein interfaces, which are less complementary electrostatically but which involve contacts that are more well-defined geometrically.…”

Section: Discussioncontrasting

confidence: 55%

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

et al. 2016

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This paper uses crystals of bovine carbonic anhydrase (CA) and its acetylated variant to examine (i) how a large negative formal charge can be accommodated in protein-protein interfaces, (ii) why lysine residues are often excluded from them, and (iii) how changes in the surface charge of a protein can alter the structure and organization of protein-protein interfaces. It demonstrates that acetylation of lysine residues on the surface of CA increases the participation of polar residues (particularly acetylated lysine) in protein-protein interfaces, and decreases the participation of nonpolar residues in those interfaces. Negatively charged residues are accommodated in protein-protein interfaces via (i) hydrogen bonds or van der Waals interactions with polar residues or (ii) salt bridges with other charged residues.The participation of acetylated lysine in protein-protein interfaces suggests that unacetylated lysine tends to be excluded from interfaces because of its positive charge, and not because of a loss in conformational entropy. Results also indicate that crystal contacts in acetylated CA become less constrained geometrically and, as a result, more closely packed (i.e., more tightly clustered spatially) than those of native CA. This study demonstrates a physical-organic approach-and a well-defined model system-for studying the role of charges in protein-protein interactions.3

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Section: Discussioncontrasting

confidence: 55%

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

et al. 2016

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“…P6-b, P6-c, and P6-e do not have this motif. Glycine and small amino acids have been suggested to promote crystallization through the reduction of surface entropy (8,14,(52)(53)(54), and the presence of GX 3 G motifs at each protein-protein interface is consistent with a tightly packed crystal. Furthermore the volume per molecular weight (Matthew's coefficient) (55 (21).…”

Section: Resultsmentioning

confidence: 99%

“…The large residue (Tyr1) at the N terminus of P6-a may also contribute to destabilization of the parallel configuration. Charged side-chains often have high conformational entropy, and they are believed to disrupt crystal-packing interfaces (8,52,53,57,58). These residues are frequently mutated to induce crystallographic order (54,59).…”

Section: Resultsmentioning

confidence: 99%

“…Hence, simultaneously achieving functional properties and the presentation of complementary intermolecular interactions that confer a targeted crystal structure can be difficult. Commonly, weak intermolecular forces stabilize crystalline ordering, and as a result, quantitative, predictive approaches to crystal design are challenging even with small molecules (7,8). In addition, within most crystals, molecules align in a nonpolar fashion (9), but polar crystalline ordering is desired for many applications, e.g., nonlinear optical materials (6).…”

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confidence: 99%

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Computational design of a protein crystal

Lanci

MacDermaid

Kang

et al. 2012

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

157

162

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Protein crystals have catalytic and materials applications and are central to efforts in structural biology and therapeutic development. Designing predetermined crystal structures can be subtle given the complexity of proteins and the noncovalent interactions that govern crystallization. De novo protein design provides an approach to engineer highly complex nanoscale molecular structures, and often the positions of atoms can be programmed with sub-Å precision. Herein, a computational approach is presented for the design of proteins that self-assemble in three dimensions to yield macroscopic crystals. A three-helix coiled-coil protein is designed de novo to form a polar, layered, three-dimensional crystal having the P6 space group, which has a "honeycomb-like" structure and hexameric channels that span the crystal. The approach involves: (i) creating an ensemble of crystalline structures consistent with the targeted symmetry; (ii) characterizing this ensemble to identify "designable" structures from minima in the sequencestructure energy landscape and designing sequences for these structures; (iii) experimentally characterizing candidate proteins. A 2.1 Å resolution X-ray crystal structure of one such designed protein exhibits sub-Å agreement [backbone root mean square deviation (rmsd)] with the computational model of the crystal. This approach to crystal design has potential applications to the de novo design of nanostructured materials and to the modification of natural proteins to facilitate X-ray crystallographic analysis.M olecular design provides powerful tools for exploring how molecular properties dictate macroscopic structure and function. One of the most precise forms of self-organization, crystallization achieves orientation and symmetry across many length scales and can be leveraged to engineer materials with well-defined molecular order (1). In addition to their central role in structure determination, molecular crystals have many applications, including nanoparticle templating (2, 3), nonlinear optical devices (4), molecular scaffolding (5), and porous frameworks (6). A predictive understanding of how to achieve self-assembled macroscale structure and desired properties remains challenging, however, particularly when large, conformationally flexible molecules are employed.Small synthetic molecules have been designed that display complementary functional groups in a manner consistent with a chosen crystal structure. Intermolecular contacts are often patterned using strong, directional interactions (e.g., hydrogen bonding, metalcoordination, and electrostatic interactions) (7). The constituent molecules, however, are typically small and synthetic, thus limiting size, shape, and functionality. Hence, simultaneously achieving functional properties and the presentation of complementary intermolecular interactions that confer a targeted crystal structure can be difficult. Commonly, weak intermolecular forces stabilize crystalline ordering, and as a result, quantitative, predictive approaches to crystal de...

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“…[26][27][28] The SER method typically replaces solvent-exposed, flexible amino acids, such as Lys, Glu, and Gln, with less flexible residues, such as Ala. It has been successfully used for several protein targets, [29][30][31] and a web-based server has been created to suggest suitable mutation sites in any given protein sequence. 30 Synthetic symmetrization involves introducing into the surface of a protein specific motifs likely to drive symmetric self-association.…”

Section: Introductionmentioning

confidence: 99%

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

Laganowsky

Zhao²,

Soriaga

et al. 2011

Protein Science

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Combining the concepts of synthetic symmetrization with the approach of engineering metal-binding sites, we have developed a new crystallization methodology termed metal-mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal-mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction-quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose-binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu 21 ), nickel (Ni 21 ), or zinc (Zn 21 ). The approach resulted in 16 new crystal structures-eight for T4L and eight for MBP-displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization-prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest.

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Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data

Cited by 132 publications

References 36 publications

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts

Computational design of a protein crystal

An approach to crystallizing proteins by metal‐mediated synthetic symmetrization

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