Crystallization, water-soluble proteins

The existence of -barrels was established for chymotrypsin at a very early stage in the now common protein crystal structure analyses. This enzyme contains two distorted six-stranded -barrels with identical topologies (Birktoft and Blow, 1972). A selection of -barrels in water-soluble proteins is given in Table I. The very abundant TIM-barrel consisting of eight parallel /1-strands was also detected rather early (Banner et al., 1975). Additional eight-stranded /1-barrels of this group are those of streptavidin (Hendrickson et al., 1989) and of the lipocalins (Newcomer et al., 1984). 

Although membrane proteins are more difficult to purify and crystallize than are water-soluble proteins, researchers using x-ray crystallographic or electron microscopic methods have determined the three-dimensional structures of more than 20 such proteins at sufficiently high resolution to discern the molecular details. As noted in Chapter 3. the structures of membrane proteins differ from those of soluble proteins with regard to the distribution of hydrophobic and hydrophilic groups. We will consider the structures of three membrane proteins in some detail. 

There were two new aspects. One was the fact that this molecule is an integral membrane protein. Structures of such molecules had not been determined before. They sit in a membrane, which is a lipid bilayer about 50 angstroms thick. This lipid bilayer is a very different environment for a protein than water is. Many of the proteins we know are water-soluble so they present a polar surface to their environment. Membrane proteins have two types of surface they present a hydrophobic surface in that part, which is inside the membrane, and a polar surface in the part that sticks out. That makes them very different to handle, to purify, to crystallize from water-soluble proteins. When we crystallize it, we have to crystallize the whole protein, both parts inside and outside of the membrane. We tried to create conditions in which both the hydrophobic surfaces and the hydrophilic surfaces of the protein were in the correct environment. That was done by coating the hydrophobic surface with so-called detergent micelles. These are molecules that have hydrophobic and hydrophilic ends. They are. similar to lipids but they don t form planar bilayers. They tend to form spherical micelles. Under the right circumstances these micelles can form a belt around the hydrophobic surface of the protein, and thus replace the lipid. This complex of the protein and the detergent has many properties like a water-soluble protein and can be crystallized as water-soluble proteins. So this was a new feature. 

One important experimental result was available, the quantitative measurement of the fraction of each secondary structural element by circular dichroism (CD) on purified lipid-protein complexes. This provided a constraint that allowed a careful evaluation of the secondary structure predictions derived from the various approaches, some of which were developed for water-soluble proteins and therefore of uncertain reliability for proteins in a lipid environment. The data from these analyses were combined using an integrated prediction method to arrive at a consensus secondary structure model for each protein. The integrated method involved 36 steps, with independent predictions at each step. The final model was based on an evaluation of the various predictions, with judicious intervention by the authors. As an aid to developing the appropriate weighting of all the data, they carried out the analysis for apoE-3 without reference to the available crystal structure (Wilson et al., 1991), then used the known structure of the HDL-binding amino-terminal domain of apoE-3 as feedback to reevaluate the weighting. 

Red-brown, water-soluble protein. Forms cubic and orrhorhombic crystals. 

FT-IR spectroscopy is particularly useful for probing the structure of membrane proteins. Until recently, a lack of adequate experimental techniques has been the reason for the poor understanchng of the secondary structure of most membrane proteins. X-ray diffraction requires high quality crystals and these are not available for many membrane proteins. Circular dichroism (CD) has been widely used for studying the conformation of water-soluble proteins, but problems arise in its use for membrane proteins. The light scattering effect may distort CD spectra and lead to substantial errors in their interpretation. In addition, the reference spectra used for the analysis of CD spectra are based on globular proteins in aqueous solution and may not be applicable to membrane proteins in the hydrophobic environment of lipid bilayers. 

Hydrophobic membrane proteins and lipids are difficult to crystallize compared to water-soluble biological molecules. Consequently, structural information on membrane components has become available at a much slower pace than on water-soluble proteins or DNA. 

Approximately one third of all proteins are tightly associated with membranes. These are much more difficult to crystallize or study by NMR than water-soluble proteins. As a consequence, there are far fewer structures of membrane proteins. Even so, those that have been determined provide insight into the manner in which polypeptide chains interact with lipid bilayers. 

The water-soluble fragment of the Rieske protein from bovine heart bci complex (ISF) was crystallized by Link et al. (30) and the structure was solved at 1.5 A resolution by Iwata et al. (9) (PDB file IRIE). 

In be complexes bci complexes of mitochondria and bacteria and b f complexes of chloroplasts), the catalytic domain of the Rieske protein corresponding to the isolated water-soluble fragments that have been crystallized is anchored to the rest of the complex (in particular, cytochrome b) by a long (37 residues in bovine heart bci complex) transmembrane helix acting as a membrane anchor (41, 42). The great length of the transmembrane helix is due to the fact that the helix stretches across the bci complex dimer and that the catalytic domain of the Rieske protein is swapped between the monomers, that is, the transmembrane helix interacts with one monomer and the catalytic domain with the other monomer. The connection between the membrane anchor and the catalytic domain is formed by a 12-residue flexible linker that allows for movement of the catalytic domain during the turnover of the enzyme (Fig. 8a see Section VII). Three different positional states of the catalytic domain of the Rieske protein have been observed in different crystal forms (Fig. 8b) (41, 42) ... 

In addition to this large movement of the Rieske protein, small but nevertheless significant conformational differences within the functional domain are observed. The structure of the functional domain of the Rieske subunit in the PGi22 crystal form showing the ci positional state is the same as that of the water soluble fragment... 

Fig. 8. (a) Structure of the full-length Rieske protein from bovine heart mitochondrial bci complex. The catalytic domain is connected to the transmembrane helix by a flexible linker, (b) Superposition of the three positional states of the catalytic domain of the Rieske protein observed in different crystal forms. The ci state is shown in white, the intermediate state in gray, and the b state in black. Cytochrome b consists of eight transmembrane helices and contains two heme centers, heme and Sh-Cytochrome c i has a water-soluble catalytic domain containing heme c i and is anchored by a C-terminal transmembrane helix. The heme groups are shown as wireframes, the iron atoms as well as the Rieske cluster in the three states as space-filling representations. 

Kerfeld, C. A., M. R. Sawaya, V. Brahmandam et al. (2003). The crystal structure of a cyanobacterial water-soluble carotenoid binding protein. Structure 11(1) 55-65. 

Insulin suspensions. When the hormone is injected as a suspension of insulin-containing particles, its dissolution and release in subcutaneous tissue are retarded (rapid, intermediate, and slow insulins). Suitable particles can be obtained by precipitation of apolar, poorly water-soluble complexes consisting of anionic insulin and cationic partners, e.g the polycationic protein protamine or the compound aminoqui-nuride (Surfen). In the presence of zinc and acetate ions, insulin crystallizes crystal size determines the rate of dissolution. Intermediate insulin preparations (NPH or isophane, lente or zinc insulin) act for 18 to 26 h, slow preparations (protamine zinc insulin, ultralente or extended zinc insulin) for up to 36 h. 

LCS Ferreira, U Schwarz, W Keck, P Charlier, O Dideberg, JM Ghuysen. Properties and crystallization of genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12. Eur J Biochem 171 11-16, 1988. 

---