Seeking Solutions in Solid-State Chemistry - Pharmaceutical Technology

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Seeking Solutions in Solid-State Chemistry


Pharmaceutical Technology Europe


Advancing protein crystallisation: microgravity effects and predictive models.

Elucidating the structure and sequence of proteins is an important task in understanding the biological properties of a protein and its potential as a therapeutic target. Producing a well-ordered crystal, particularly for proteins, which can be studied through crystallography, however, is not an easy task. Recent research involves examining the effects of microgravity on protein crystallisation and a computational model for protein elucidation.

Protein crystallisation and microgravity effects

The Centre for the Advancement of Science in Space (CASIS), manager of the International Space Station (ISS) US National Laboratory, is collaborating with Merck & Co. to conduct research on protein crystallisation on board the ISS in 2013. The research will examine the effect on protein crystallisation using microgravity.

In July, CASIS announced its first request for proposals (RFP) focused on advancing protein crystallisation using microgravity. Additionally, in early September 2012, CASIS announced an RFP focused on materials testing in the extreme environment of space. Proposals for this RFP will be accepted until 24 Oct. 2012. The final agreement with Merck is dependent on approval by CASIS' evaluation and prioritisation process, a requirement for all ISS projects. If approved, the research will begin in mid-2013.

"We at Merck are excited to work with CASIS and explore the microgravity effects on several bioprocessing applications within the unique environment of the ISS National Lab," said Paul Reichert, chemistry research fellow at Merck Research Laboratories, in a September CASIS press release.

CASIS is the nonprofit organisation promoting and managing research on board the ISS US National Laboratory, which includes a solicitation for proposals in relation to advancing protein crystallisation using microgravity. The RFP seeks to identify projects within the field of crystallography that CASIS will support through grant funding, facilitation of service provider partnerships, and flight coordination to and from the ISS. Crystallography is the technique used to determine three-dimensional structures of protein molecules. Protein crystallisation, when performed in space, may produce large, better-organised crystals, thereby allowing for more focused drug development. CASIS believes that its RFP will lead to the production of better crystals in the microgravity environment than can be grown on Earth.

"CASIS has evaluated research performed to date in the life sciences and believes it is time to formally test the promising hypothesis that microgravity may produce greater internal order in protein-crystal growth," said CASIS acting Chief Scientist Timothy Yeatman, in a 26-June 2012, CASIS press release. "This could potentially lead to sharper resolution of crystals and their cognate proteins, which could produce more effective drugs for cancer and other debilitating human diseases."

In 2005, the US Congress designated the US portion of the ISS as the nation's newest national laboratory to maximise its use for improving life on Earth, promoting collaboration among diverse users, and advancing science, technology, engineering and mathematics education. The laboratory environment is available for use by other US government agencies and by academic and private institutions to provide access to the permanent microgravity setting, vantage point in low-earth orbit and varied environments of space.

Computational approaches for protein elucidation

Determining the structure and sequence of proteins is an important part of understanding the protein's biological properties and potential utility as a drug. Designing predetermined crystal structures, however, can be subtle given the complexity of proteins and the noncovalent interactions that govern crystallisation (1). Researchers at the University of Pennsylvania recently reported on a computational approach for the design or proteins that self-assemble in three dimensions to yield macroscopic crystals (1).

"People have designed crystals out of smaller, much less complex molecules than proteins, but protein design is much more subtle," said Jeffrey G Saven, associate professor of chemistry and biological and theoretical physical chemistry at the University of Pennsylvania, in a university press release. Saven conducted the research and recently reported on its results (1). Protein crystals are attractive as a nano-scale building material because their properties, particularly their exterior surfaces, are highly customisable, according to the university release.

The researchers targeted a crystal built using a relatively small protein containing a sequence of 26 amino acid positions. The researchers assigned specific amino acids to eight of the positions, but with 18 different types of amino acid to choose from for each of the remaining 18 slots, the algorithm addressed well more than 1022 potential combinations. The researchers accounted for other characteristics, such as the spacing between proteins and their orientation with respect to one another, increasing the variables being considered, according to the release.

"We worked on synthesising both of those steps, doing the characterisation of structure and the sequence at the same time," said Saven said in the university release. "As we move through this process, we eliminate things that will never work, such as proteins where atoms overlap in space or where amino acids don't fit into a given site. At the same time, we identify proteins that, as you vary the structure, are likely to yield a crystal."

Specifically, the research used a three-helix coiled-coil protein designed de novo to form a polar, layered, three-dimensional crystal having the P6 space group, which had a "honeycomb-like" structure and hexameric channels that spanned the crystal (1). The approach involved creating an ensemble of crystalline structures consistent with the targeted symmetry, characterising this ensemble to identify "designable" structures from minima in the sequence–structure energy landscape and designing sequences for these structures, and experimentally characterising candidate proteins. 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.

The target crystal the researchers produced is a proof of concept. "There's still much we don't know about the interactions that govern crystallisation," Saven said, in the university release. "With this technique, we can explore what those interactions are or how we might take an existing protein and engineer those interactions so we get much better structures."

Source

1. J.G. Saven et al, PNAS, 109 (19), 7304–7309 (2012).

Patricia Van Arnum is executive editor of Pharmaceutical Technology Europe.


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