Structural Biochemistry/Overcoming Challenges of Protein Crystallography

From Wikibooks, open books for an open world
Jump to: navigation, search

Because membrane proteins continue to be one of the most difficult targets for structural biologists because all cells have a lipid-bilayer with proteins embedded through the lipid bilayer and in addition to those proteins, there are often domains outside the membrane. These proteins have been thoroughly studied and they are involved in an array of processes such as respiration, photosynthesis, signal transduction and molecular transport, etc.

Only a few of the structures of the 20-30% of the proteomes represented by membrane proteins have been solved through either X-ray Crystallography or NMR. Only about a little over 1% of about over 50,000 entries about proteins in the Protein Data Bank are information pertaining the membrane proteins. The reason for this small fraction of membrane protein information, it can be concluded that membrane proteins are difficult to study. Because of the membrane proteins have a hydrophobic surface, this property enables them to be extracted from the cell membrane only with detergents used alongside. These membrane proteins are also often very flexible and unstable and this is an important challenge to recognize because it is then difficult at a variety of levels (expression/solubilization/crystallization/data collection/purification/structure solution).

Expression and Purification[edit]

There are some known structures of membrane proteins that have either been synthesized chemically (for short peptides) or purified (from natural sources) or even produced recombinantly. For example, Lactococcus lactis, Pichia pastoris, Saccharomyces cerevisiae and Escherichia Coli have all been expressed in insect cells and in mammalian cell lines. More specifically, the production of E.Coli is very rapid, inexpensive and easy to utilize with screens and this therefore influences the success of an expression system. But for eukaryotic proteins, a different system has to be used- a eukaryotic system is required in order for expression. First, membrane proteins can only fold correctly if they have been targeted by the host cell membrane. Next, the nature of lipids and composition of the lipids in which the membrane proteins are embedded throughout will vary throughout the systems. Therefore, the affects of the stability of the protein is resulted from the nature of the lipids can consequently result to the likelihood of crystallization.Thirdly, Eukaryotic proteins may undergo glycosylation and other modifications (after translation). 17 of the 39 unique eukaryotic membrane proteins were produced using recombinant methods while the other nine were from yeast systems; there were also four in insect cells and another four found in E. Coli.

Detergents[edit]

Detergents have an important role in extracting membrane proteins from the host cell membrane because detergents are able to surround the hydrophobic surfaces of the proteins and this allows for solubilization to occur. Because of this crucial step, it is also important to carefully choose a detergent for this purification process. Detergents are tested many times and are tested for it's ability to extract the largest amount of soluble, active and stable protein. But there are examples of detergents that are efficient in terms of extracting proteins from the membrane but those detergents are not as efficient at guaranteeing solubilized membrane proteins i.e. FOS-Choline. Therefore, there must be detergents that need to satisfy many properties; Dodecyl Maltoside (DDM) is a particular detergent that is used to extract membrane proteins from the lipid bilayer and it can give solubilized membrane proteins in a stable manner at a relatively cheap cost.

There has been many progress in the development of the different methods in assessing the expressions and purifications of membrane proteins. One technique of following the protein during purification, that was introduced by Professor Jan-Wilem De Gier and Eric Gouaux, was the ability of utilizing the cleavage of a GFP (green fluorescent protein) with a His-tag fused to the C-terminus of this protein. In the example of E. Coli expression systems, this system became successful under the conditions that the target membrane protein had a cytoplasmic C-terminus; this was because the green fluorescent protein can only become fluorescent in the cytoplasm and can only be folded correctly in the cytoplasm. Therefore, the highest expression yields for large-scale purification was shown through the prokaryotic and eukaryotic green fluorescent protein fusino proteins that were expressed through S. Cerevisiae and E. Coli. Many times a GFP fusion protein may not need preceding purification and this can be clearly shown through gel fluorescence analysis and fluorescence size-exclusion chromatography.

Another technique in screening many constructs and conditions for expression and solubilization of membrane proteins has been explained and it was initially explained for proteins with small-scale purification, more specifically for proteins in a 96-well format. But from its initial explanation, it has been altered in order to recognize protein expression within colonies; this technique is achieved through the colony filtration blot method expression in induced after colonies are blotted onto the membrane and then the cells are lysed with the variety of test detergents. Once these detergent solubilized proteins are then filtered throughout the membranes they are detected with antibodies against the tag. Finding conditions in which the membrane protein is more stable could potentially precede improvement in crystallization but membrane proteins tend to be in an unstable condition in detergent micelles. Oftentimes lipids are added in order to maintain a stable solubilized sample; methods of gel filtration, electron microscopy or ultracentrifugation are utilized in order to monitor the aggregation states of the material when different buffers and detergent conditions are screened for. Thermal stability has also been studied as a means of monitoring the state of a protein. Many research has been furthered by the analysis of the fluorescence of a covalent bond to a dye attached to an accessible cysteine residue. Also, there has been recent studies on improved stability on� ß1-adrenergic receptor with the using point mutations and testing the resulting mutants for activity.

Crystallization[edit]

The process of protein crystallization happen through the process of multiple tests performed on a large number of reagents that could potentially be utilized. But these initial crystallization conditions are not sufficient enough to determine whether or not it's fully optimal until well-diffracting crystals are obtained. Throughout the past decade, necessary routine screenings for crystals have been achieved with 100 nl drops and even though there are many 96-well sparse matrix screening systems that have been made available for soluble proteins, these al contain many specific properties in which proteins are not very likely to crystallize in. Therefore, there have been specifically designed crystallization screens designed by the Iwata group that have been prepared in order to optimize protein crystallization for membrane proteins. After much evidence, it has been proved that it's an important process to screen the detergent during crystallization. Crystal quality can also be improved with the addition of detergents that are present in the crystallization drop. Therefore, the proteins molecules can be ordered in a manner so that they can form crystals because of the ability of the three-dimensional continuous lipidic phases; these type of phases allow for membrane proteins to freely diffuse in the lipid rather than having the membrane proteins being enclosed within detergent micelles.

Data Collection and Structure Solution[edit]

Data collection on soluble protein crystals have now become a routine with the crystals set on the sample changer and the automatic data collection. This situation has become difficult for membrane protein crystals because the crystals from membrane proteins usually contain a high solvent content due to the detergent micelle (the detergent micelle will surround the hydrophobic portion of the protein). Therefore, the crystals are then difficult to handle because of their fragility with a very low quality of the crystal. This type of data collection would then result in large number of crystals needing to be screened. But because of synchotron beamlines of automatic sample changes, the previously mentioned situation has been addressed and enables many crystals to be screened efficiently and quickly. Datasets are collected from these beamlines and these beamlines collect it from the small crystals while also collecting data from the well diffracting regions. Datasets are also collected from the lengths of the crystals even though there are individual portions that suffer from radiation damage. Large improvements in the phases have resulted in solvent flattening due to the high solvent substance of membrane proteins.

Conclusion[edit]

There has been much evidence that state that the structure solution of membrane proteins still retains many specific challenges in comparison to soluble proteins. But a variety of advances have been made in order to engineer proteins that are able to be crystallized and for those proteins to be stabilized. Within the next few years, it is speculated that there will be a rapid growth in the amount of membrane protein structures that will be solved especially for eukaryotic membrane proteins. Not only will these types of discoveries help with our understanding of protein folds and protein function in the membrane but it will also provide a lot of information on the potential design for future novel drugs.