Fusion to the TELSAM protein polymer dramatically improves the speed of target protein crystallization by stabilizing weak crystal contacts

We extend investigation into the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We tested various numbers of the target protein fused per turn of the TELSAM helical polymer and various TELSAM–target connection strategies. We provide definitive evidence that: 1. A TELSAM–target protein fusion can crystallize more rapidly than the same target protein alone, 2. TELSAM–target protein fusions can form well-ordered, diffracting crystals using either flexible or rigid TELSAM–target linkers, 3. Well-ordered crystals can be obtained when either 2 or 6 copies of the target protein are presented per turn of the TELSAM helical polymer, 4. The TELSAM polymers themselves need not directly contact one another in the crystal lattice, and 5. Fusion to TELSAM polymer confers immense avidity to stabilize exquisitely weak inter-target protein crystal contacts. We report features of TELSAM-target protein crystals and outline future work needed to define the requirements for reliably obtaining optimal crystals of TELSAM–target protein fusions.

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The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14027861 ◽

2015 ◽

Vol 71 (2) ◽

pp. 157-162 ◽

Cited By ~ 6

Author(s):

Yancho D. Devedjiev

Keyword(s):

Protein Crystallization ◽

Protein Structures ◽

Physicochemical Characteristics ◽

Molecular Shape ◽

Side Chain ◽

Static Disorder ◽

Relative Contribution ◽

Crystal Contacts ◽

Essential Prerequisite

Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d< 1.5 Å) reveals that in contrast to current views, static disorder and high side-chain entropy are common in the crystal contact area. These observations challenge the validity of the theory that presumes that the occurrence of well ordered patches of side chains at the surface is an essential prerequisite for a successful crystallization event. The present paper provides evidence in support of the approach for understanding protein crystallization as a process dependent on multiple factors, each with its relative contribution, rather than a phenomenon driven by a few dominant physicochemical characteristics. The role of the molecular shape as a factor in the crystallization of proteins by surface mutagenesis is discussed.

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The potential of hexatungstotellurate(VI) to induce a significant entropic gain during protein crystallization

IUCrJ ◽

10.1107/s2052252517012349 ◽

2017 ◽

Vol 4 (6) ◽

pp. 734-740 ◽

Cited By ~ 21

Author(s):

Christian Molitor ◽

Aleksandar Bijelic ◽

Annette Rompel

Keyword(s):

Protein Crystallization ◽

Accessible Surface Area ◽

Solvent Accessible Surface Area ◽

Limiting Factor ◽

Hydration Shells ◽

Starting Point ◽

Crystal Contacts ◽

Accessible Surface ◽

Solvent Accessible Surface

The limiting factor in protein crystallography is still the production of high-quality crystals. In this regard, the authors have recently introduced hexatungstotellurate(VI) (TEW) as a new crystallization additive, which proved to be successful within the liquid–liquid phase separation (LLPS) zone. Presented here are comparative crystal structure analyses revealing that protein–TEW binding not only induces and stabilizes crystal contacts, but also exhibits a significant impact on the solvent-driven crystallization entropy, which is the driving force for the crystallization process. Upon the formation of TEW-mediated protein–protein contacts, the release of water molecules from the hydration shells of both molecules,i.e.TEW and the protein, causes a reduced solvent-accessible surface area, leading to a significant gain in solvent entropy. Based on the crystal structures of aurone synthase (in the presence and absence of TEW), insights have also been provided into the formation of a metastable LLPS, which is caused by the formation of protein clusters, representing an ideal starting point in protein crystallization. The results strongly encourage the classification of TEW as a valuable crystallization additive.

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Best architecture of protein crystal. Database of protein-polymer interactions showing a unique role of PEG in protein crystallization

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273316096352 ◽

2016 ◽

Vol 72 (a1) ◽

pp. s241-s241 ◽

Cited By ~ 1

Author(s):

Jindřich Hašek ◽

Tereza Skálová ◽

Petr Kolenko ◽

Jarmila Dušková ◽

Tomáš Koval ◽

...

Keyword(s):

Protein Crystallization ◽

Protein Crystal ◽

Unique Role ◽

Protein Polymer ◽

Polymer Interactions

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Controlling Protein Crystallization by Free Energy Guided Design of Interactions at Crystal Contacts

Crystals ◽

10.3390/cryst11060588 ◽

2021 ◽

Vol 11 (6) ◽

pp. 588

Author(s):

Johannes Hermann ◽

Daniel Bischoff ◽

Phillip Grob ◽

Robert Janowski ◽

Dariusch Hekmat ◽

...

Keyword(s):

Free Energy ◽

Protein Interactions ◽

Computational Study ◽

Protein Crystallization ◽

Lactobacillus Brevis ◽

Protein Crystal ◽

Crystal Formation ◽

Contact Stability ◽

Crystal Contacts ◽

Energy Simulations

Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis (LbADH) as a model system, we probed in our combined experimental and computational study the effect of residue substitutions at the protein crystal contacts on the crystallizability and the contact stability. Increased or decreased contact stability was calculated using molecular dynamics (MD) free energy simulations and showed excellent qualitative correlation with experimentally determined increased or decreased crystallizability. The MD simulations allowed us to trace back the changes to their physical origins at the atomic level. Engineered charge–charge interactions as well as engineered hydrophobic effects could be characterized and were found to improve crystallizability. For example, the simulations revealed a redesigning of a water mediated electrostatic interaction (“wet contact”) into a water depleted hydrophobic effect (“dry contact”) and the optimization of a weak hydrogen bonding contact towards a strong one. These findings explained the experimentally found improved crystallizability. Our study emphasizes that it is difficult to derive simple rules for engineering crystallizability but that free energy simulations could be a very useful tool for understanding the contribution of crystal contacts for stability and furthermore could help guide protein engineering strategies to enhance crystallization for technical purposes.

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Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714025061 ◽

2015 ◽

Vol 71 (3) ◽

pp. 427-441 ◽

Cited By ~ 3

Author(s):

Mark Stauber ◽

Jean Jakoncic ◽

Jacob Berger ◽

Jerome M. Karp ◽

Ariel Axelbaum ◽

...

Keyword(s):

Small Molecule ◽

Protein Crystallization ◽

Protein Structures ◽

Preferential Interaction ◽

Crystal Contacts ◽

Chiral Interactions

Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using theRandSenantiomers as well as with a racemicRSmixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

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Split green fluorescent protein as a modular binding partner for protein crystallization

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913024608 ◽

2013 ◽

Vol 69 (12) ◽

pp. 2513-2523 ◽

Cited By ~ 17

Author(s):

Hau B. Nguyen ◽

Li-Wei Hung ◽

Todd O. Yeates ◽

Thomas C. Terwilliger ◽

Geoffrey S. Waldo

Keyword(s):

Crystal Structure ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Protein Crystallization ◽

Target Protein ◽

Surface Loop ◽

Binding Partner ◽

Green Fluorescent ◽

The Way

A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization.

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The microtubule associated protein (MAP) tau forms a new class of triple-stranded left-hand helical fibrous protein polymer

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123106 ◽

1992 ◽

Vol 50 (1) ◽

pp. 540-541

Author(s):

G. C. Ruben ◽

K. Iqbal ◽

I. Grundke-Iqbal ◽

H. Wisniewski ◽

T. L. Ciardelli ◽

...

Keyword(s):

Axial Ratio ◽

Normal Structure ◽

Paired Helical Filaments ◽

Left Hand ◽

New Class ◽

Protein Polymer ◽

Fibrous Protein ◽

Protein Map ◽

Neurotransmitter Transport ◽

Alzheimer Neurofibrillary Tangles

In neurons, the microtubule associated protein, tau, is found in the axons. Tau stabilizes the microtubules required for neurotransmitter transport to the axonal terminal. Since tau has been found in both Alzheimer neurofibrillary tangles (NFT) and in paired helical filaments (PHF), the study of tau's normal structure had to preceed TEM studies of NFT and PHF. The structure of tau was first studied by ultracentrifugation. This work suggested that it was a rod shaped molecule with an axial ratio of 20:1. More recently, paraciystals of phosphorylated and nonphosphoiylated tau have been reported. Phosphorylated tau was 90-95 nm in length and 3-6 nm in diameter where as nonphosphorylated tau was 69-75 nm in length. A shorter length of 30 nm was reported for undamaged tau indicating that it is an extremely flexible molecule. Tau was also studied in relation to microtubules, and its length was found to be 56.1±14.1 nm.

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