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It was necessary to design a system with radiation-hardened circuits so the missiles could go through these nuclear bomb clouds. One key feature of these circuits was the need for stability in the conductivity of the substrate. Silicon was the technology of choice, but it suffered when exposed to large amounts of radiation.

My colleague, Harold Manasevit had the idea of growing silicon on a sapphire substrate, which was an insulator from radiation and infinitely stable. So he developed a technology called silicon on sapphire, or SOS, which was used in the Minuteman Missiles.

Mod-01 Lec-14 Crystal growth-Single crystals

One set is from the proceedings of a Symposium held in August to celebrate 50 years of progress in the field of crystal growth. The first section of this book contains several articles which describe some of the early history of crystal growth prior to the electronics revolution, and upon which modern crystal growth science and technology is based. This is followed by a special article by Prof. Sunagawa which provides some insight into how the successful Japanese crystal growth industry developed. The next section deals with crystal growth fundamentals including concepts of solute distribution, interface kinetics, constitutional supercooling, morphological stability and the growth of dendrites.

These articles were written by some of the most famous theorists and crystal growers working in the field. They will provide future research workers with valuable insight into how these pioneering discoveries were made, and show how their own research and future devices will be based upon these developments. The recent modest increase in the rate of determining membrane-protein structures has been facilitated by improvements in the areas of membrane-protein molecular biology and biochemistry, and through technical advances in synchrotron X-ray beamlines for crystallography, high-field nuclear magnetic resonance NMR and high-resolution electron microscopy.

The availability of sequenced genomes spanning a broad range of species has vastly improved searches for structural homologs and the prediction of previously unknown membrane proteins.

1. Introduction

These factors have converged to help set the stage for the determination of membrane-protein structures rapidly and on a large scale. In recent years a number of consortia, bringing together researchers from a variety of academic and research institutions [ 5 — 7 ]; have been established to address and execute the goals of structural genomics - that is, to dramatically increase the database of known protein structures by developing and applying methodologies to determine them as rapidly and cost-effectively as possible.

Structural genomics of membrane proteins | Genome Biology | Full Text

To date, however, only one group Mycobacterium tuberculosis Membrane Protein Structural Genomics [ 8 ] has taken on as its primary mission the high-throughput determination of membrane-protein structures. While the efforts of this group are ongoing, substantial progress has already been made in the construction of expression vectors on a large scale.

This article provides an overview of the factors essential for the determination of membrane-protein structures in high-throughput fashion and the progress that has been made so far in these areas. The key issues that arise for a researcher who wishes to determine the structure of membrane proteins at the atomic level are: how to produce sufficient protein, and once produced how to solubilize and purify the protein; then, how to crystallize the protein, or whether instead to study it in solution; and finally, how to scale up such methods for high-throughput structure determination.

High-resolution structure-determination efforts typically require milligram quantities of proteins. Overexpression of prokaryotic genes in bacterial vectors currently provides the most direct and productive route to fulfilling this need [ 9 , 10 ]. Studies on genes with introns will require full-length cDNAs derived from mRNA libraries, and this represents another degree of complexity. Groups such as the Mammalian Gene Collection MGC [ 11 ], for example, have created resources for the production and distribution of full-length human genes [ 12 ].

Prokaryotic genomes are also logical choices as target genomes for membrane-protein structural genomics efforts [ 13 ]. The initial goals of these efforts will be to clone, overexpress and purify the known and putative membrane proteins of their selected genomes. Potential membrane-protein targets can be identified from functional studies or on the basis of knowledge from previously characterized homologous genes.

In many instances, however, homology-based predictions of protein type and function will not be possible. For these proteins, assignment of putative membrane-protein status will have to be based on predictions of transmembrane segments using the many bioinformatic tools now available for example, see [ 14 ]. Ideally, all successfully expressed and solubilized target membrane proteins should be distributed to X-ray and electron crystallography groups, and appropriate protein samples to NMR spectroscopy teams, for simultaneous efforts at structure determination and maximization of the likelihood that rapid progress will be made.

Once a membrane protein has been prioritized, by whatever means, for structure determination, the next step must be to overexpress the protein in a way that allows significant quantities to be isolated and purified for further study. The majority of structural genomics consortia are pursuing high-throughput protein expression through constructs expressed in Escherichia coli.

Expression in E. It has clear advantages currently with respect to cost per gene expressed, the variety of specialized expression vectors available and the well-developed methods for labeling target proteins for NMR and X-ray diffraction studies [ 9 , 10 ].

Expression vectors based on promoters used by T7 RNA polymerase are in widespread use for the overexpression of soluble proteins among the various consortia [ 9 , 10 ]. It also appears that for the immediate future this class of vectors will be favored by research groups overexpressing membrane proteins. One concern, especially with respect to the overexpression of membrane proteins, is the effect of target-protein expression levels in the uninduced state 'leakiness' on host-cell health, and subsequently on the ability to overexpress properly folded proteins at high levels.

Vectors with promoters less prone to leakage expression may have to be sought for the successful overexpression of certain membrane proteins. Purification of overexpressed protein is greatly simplified and idealized for high-throughput studies through the use of constructs in which the target gene is fused to an affinity tag, whereby the tag can be placed at either the amino- or the carboxy-terminal end of the target protein, with a number of options in construct design.

Examples of tags include glutathione S -transferase, maltose-binding protein and polyhistidine.

By virtue of their ease of use, polyhistidine tags have seen the broadest application [ 9 , 10 ]. Although there are indications that amino-terminal polyhistidine-tag fusion proteins may have a better expression record with respect to membrane proteins [ 8 ], in our view the performance of both amino- and carboxy-terminal tagged constructs should be evaluated on a case-by-case basis with respect to target-protein overexpression, solubility, and crystallizability.

To facilitate the subsequent removal of affinity tags, protease recognition sites can also be incorporated into the constructs; and in the case of membrane proteins it is desirable that these sites support the use of detergent-resistant proteases, so as to be compatible with detergent-based purification procedures. Structure-determination efforts on human gene products have been limited, because of difficulties in obtaining high expression levels of protein.

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Many human genes will probably require some form of eukaryotic expression vector for successful overexpression. Numerous yeast, insect and mammalian cell lines could potentially serve in this capacity [ 13 ]; the development of eukaryotic expression methodologies tailored for high-throughput applications, however, is still in the nascent stages. The choice of host cells for overexpression of a given protein will depend on various factors, such as the source of the original gene, the protein's fold complexity and the potential need for folding partners, and requirements for post-translational modification.

As discussed earlier, the use of E. Although there are a number of strains that have been used to express membrane proteins, BL21 DE3 and a derivative of BL21 optimized for membrane-protein expression designated C43 appear to be best suited for the task. Strain variant C43 grows more slowly than BL21, and in doing so may provide more time for the host cell to deal properly with higher than normal levels of membrane-protein expression [ 15 ].

Expression of both soluble and membrane proteins in a given bacterial strain can be quite sensitive to post-induction incubation temperature. The amount of overexpressed target membrane protein localized within lipid bilayers may be increased, and the occurrence of inclusion bodies containing aggregated protein reduced, by lowering incubation temperatures following induction. Although not as practical for high-throughput purposes as bacterial expression systems, certain eukaryotic target proteins, either single polypeptides or those of multiple subunit complexes, may require 'higher' cell types to achieve adequate expression.

Potential drawbacks to the use of eukaryotic cell types can derive from difficulties in protein isolation and yield, longer doubling times, and cost. Dealing with post-translational modifications, such as the removal of glycosylation usually required for success in crystallization, can be particularly challenging and may require inhibition of the host cell's glycosylation pathway during expression, or modification of the construct sequence, or treatment of expressed proteins with glycosidases [ 16 ].

Although still an evolving technology, cell-free expression systems offer an alternative methodology for the overexpression of proteins [ 17 — 19 ]. In such a system, essential protein expression machinery is obtained from cell lysates, which can be isolated and prepared in-house or obtained from commercial sources. Commercial systems are presently available with an advertised capability of producing mg of protein from 30 ml of reaction mixture over a hour time period [ 20 ]. Clearly this level of expression is well suited for high-throughput structural studies.

For membrane proteins, however, expression away from lipid bilayer environments can, understandably, result in problems with protein folding and solubility.

1951: Development of Zone Refining

Supplementation of the reaction mixtures with detergents and lipids may provide a means of extending the utility of this approach to membrane proteins. Once sufficiently high level expression of the target membrane protein has been established, preferably in the host-cell membrane, the next step is to determine the detergents best-suited for solubilization and subsequent purification. A wide variety of detergents suitable for membrane-protein solubilization are currently available see, for example, [ 21 ]. Some of the most popular detergent families include the alkyl glucosides and maltosides, polyoxyethylenes, alkyldimethylamine oxides, and cholate derivates.

Experience has shown that the detergent selected for membrane extraction may not be the detergent of choice for crystallization.

Broadly speaking, both the length of the detergent's hydrocarbon chain hydrophobic domain and the size of its polar head group hydrophilic domain are major factors affecting the stability of the solubilized protein - longer chain lengths and larger head groups are generally more favorable for the stability of the protein.

When necessary, solubilizing detergents can be exchanged for other detergents through dialysis, or while the target protein is bound to chromatographic media. Some factors that need to be evaluated in choosing a detergent at the solubilization stage may be extraction yield, stability of the solubilized protein, and cost.

A particularly important criterion in selecting a detergent is its effect on a protein's structure and function. Certain detergents, particularly ionic ones, can denature membrane proteins, even when used at relatively low concentrations. Undesirable outcomes can involve varying degrees of denaturation, separation of subunits from multimeric or multisubunit complexes, and aggregation [ 13 ]. Evaluation of a detergent's effect on target-protein stability can provide a relatively quick means of assessing a detergent's suitability. One simple but effective test we have used involves solubilizing target proteins in candidate detergents and storing the mixtures overnight at room temperature.

The various preparations can then be quickly evaluated to determine whether or not the protein has precipitated.

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Those solubilized proteins appearing stable can be examined more closely to determine the extent of homogeneity. Dynamic light scattering is another approach that can provide much of the same information about particle size as molecular-sieve chromatography, but more rapidly [ 23 ]. A form of NMR spectroscopy, heteronuclear single quantum correlation HSQC , can also be used as a screening tool for the rapid assessment of target-protein quality [ 22 ]. If the function of a target protein is known or confidently predicted, functional assays should ideally be used to ensure that the protein is fully active in the candidate detergent.

As in the case of high-throughput structure-determination efforts for soluble proteins, the process of purifying overexpressed membrane proteins has been substantially streamlined through the use of affinity tags. When coupled to the output of optimized host-cell systems, milligram quantities of relatively pure protein can be obtained following a single chromatographic step [ 13 ].

In many instances the target protein will already be sufficiently pure at this stage to begin structure-determination efforts.

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During this phase of purification it is also appropriate to address the possibility of whether the solubilizing detergent used is suitable for crystallization and for maintaining a monodisperse solution when the protein is highly concentrated. The effects of alternative detergents can be investigated by exchanging detergents while the target protein is bound to the affinity column.

Should the affinity-column-purified sample require further cleanup, use of molecular-sieve chromatography is usually sufficient to remove minor contaminants and aggregates. On those occasions when residual contaminants cannot be isolated from the target protein on the basis of molecular weight differences, an alternative additional chromatographic step, such as ion-exchange chromatography, may be necessary [ 10 ].