Protein structure by x-ray crystallography

Thiol groups have a high affinity for mercury ions including organic mercury derivatives, which are widely used in the determination of protein structures by X-ray crystallography (Section F). Titration of SH groups in proteins is often accomplished with... 

The accumulation of E-Tyr-AMP in the absence of tRNA and the stability of the complex were crucial for the initial success of protein engineering. These factors allow active-site titration and pre- steady state kinetics. Further, the longterm stability of E Tyr-AMP enables the direct solution of its structure by x-ray crystallography. 

The primary method for determining protein structure is x-ray crystallography. Unfortunately, the receptors most frequently targeted by the pharmaceutical industry are embedded in cell membranes. The cell membrane plays a vital role in determining the overall shape of a membrane-bound receptor. Crystallization is performed in the absence of membrane lipids, so x-ray information is not representative of the true receptor structure. Therefore, reliable structural information on receptors is difficult to obtain. 

The most informative analysis of ecdysteroid/receptor interaction would be by X-ray crystallography. Although it has been possible to crystallise the LBDs of several nuclear receptors and to determine their structures by X-ray crystallography (ER [208], PR [209], RAR [210], RXR [211], TR [212], USP [213] and VDR [214]), it has not yet been possible to do this for any EcR protein. This probably derives from the fact that EcR only binds ligand with high affinity when it is complexed with USP (RXR), necessitating the co-crystallisation of ligand + EcR LBD + USP LBD. Ultimately it should be possible to achieve this. In the meantime, two... 

As discussed previously, crystallization of multicomponent proteins is indispensable not only for determining the structure by X-ray crystallography but also for obtaining stable preparations at the high level of purity required for chemical composition and functional analyses. However, crystallization conditions are influenced by a multitude of factors, some of which will be described below. 

As with any protein simulation, the nature and limitations of the structural solutions for proteins provided by X-ray crystallography should always be borne in mind [125]. One obvious point is that hydrogen atoms are generally not observed because of their low electron density (neutron diffraction experiments can be useful to overcome this problem), and so it can be difficult to assign protonation states unambiguously, and to decide between possible rotamers or tautomers. This, and other factors such as model bias (for example in a molecular replacement solution), or simple error in construction of a model, may lead to the structural model being incomplete or incorrect in some places. 

The stability of the acyl-enzyme complexes formed with C freundii class C beta-lactamase allowed solution of their structures by X-ray crystallography (Fig. 8). No significant changes in protein structure occurred, except that the side chain of Aspl23 moved to accommodate... 

The crystals of several clostridial ferredoxins are shown in Figure 2. It indicates some of the problems involved in the determination of the structure of the protein. The crystalline forms from different clostridial species differ, but all are very small crystals. The largest ones I have seen are these from Clostridium acidi-uiici, but even these are very small as far as a crystallographer is concerned, and unfortunately, they are multiple crystals. There has been very little work done on the structure by x-ray crystallography. One problem is the small size of the crystals the second diflSculty is the fact that it has not been possible to obtain any heavy metal replacement for these proteins. However, Jensen has gotten some information on clostridial type ferredoxin that I will discuss a httle later. The plant type ferredoxin has not been obtained in crystalline form that is suitable for any x-ray work. 

Numerous reports have been published about the three-dimensional structure of proteins determined by X-ray crystallography and/or NMR, and by computational chemical calculation from the results of amino acid sequencing. An empirical approach to identifying catalytic sites, the location of metal ion and carbohydrate binding sites, and folding and unfolding, has been studied with molecular dynamics simulations. Once the binding site of a protein, the structure of the protein-small molecule interaction, is... 

Even more information may be obtained by the. separate determination of A// and AS from differential scanning calorimetry, from the binding of 8-anilino-l-naphthalenesul-fonic acid, a fluorescent dye which preferentially binds to the molten globule state, and from proton exchange rates determined by NMR spectroscopy. Ultimately, all of these methods are required for a complete characterization of the protein. The final test remains the determination of the three-dimensional structure by X-ray crystallography or NMR. For a soluble, stable, folded protein, this should generally be possible. 

In many areas of chemistry, chemical methods of structure proof have given way to spectroscopic methods (Chapter 14) and analysis by mass spectrometry. In the field of carbohydrate chemistry, however, chemical reactions play an important part in the elucidation of the structures of unknown carbohydrates. The reason for this is that many proteins and lipids contain carbohydrates that have very complex structures, and these do not yield easily to spectroscopic methods. Also, since it is notoriously difficult to crystallize many carbohydrates, proof of structure by X-ray crystallography is often not possible. In this section we will discuss methods for determining determine the structure of a monosaccharide by chemical methods. Several synthetic reactions can be used to convert one monosaccharide into another. If the structure of a related monosaccharide is known, and the mechanism of the reaction that interconverts them is well understood, then the structure of the unknown monosaccharide can be established. 

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