Protein crystallization amino acid structures

As already pointed out in Part III, Chapter 19, in the crystal structures of proteins the amino acid main-chain and side-chain atoms at the periphery of the molecule display larger thermal motion (associated with larger B-factors [A2]) than those in the interior. Since the water of hydration molecules are bound primarily to these peripheral protein atoms, they display similar, and in general even more excessive thermal motion. As a consequence, the interpretation of the hydration water structure can be ambiguous, as outlined in Box 23.1. 

Although the positions of all E dimers in the outer shell of the particle were known, a precise interpretation of the density contributed by the M protein was not possible. This was due to the lack of detailed information concerning the C-terminal 101 amino acids of the E protein that were missing from the crystal structure. These residues form the stalk region, the transmembrane domain, and the NSl signal sequence. Approximately 52 residues would compose the stalk and are found in a shell of density in which the short M protein (37 amino acids outside of the membrane) would also be predicted to be found. Together, the M and the E proteins completely cover the lipid bilayer so that there is no exposed membrane in the dengue particle. 

Fig. 1. Superposition of three crystal structures of cAMP-dependent protein kinase that show the protein in a closed conformation (straight line), in an intermediate conformation (dashed line), and in an open conformation (broken line). The structures were superimposed on the large lobe. In three locations, arrows identify corresponding amino acid positions in the small lobe.

It can be seen from Table 2 that the intrinsic values of the pK s are close to the model compound value that we use for Cys(8.3), and that interactions with surrounding titratable residues are responsible for the final apparent values of the ionization constants. It can also be seen that the best agreement with the experimental value is obtained for the YPT structure suplemented with the 27 N-terminal amino acids, although both the original YPT structure and the one with the crystal water molecule give values close to the experimentally determined one. Minimization, however, makes the agreement worse, probably because it w s done without the presence of any solvent molecules, which are important for the residues on the surface of the protein. For the YTS structure, which refers to the protein crystallized with an SO4 ion, the results with and without the ion included in the calculations, arc far from the experimental value. This may indicate that con-... 

An effective method for localizing causes of redox potentials is to plot the total backbone and side chain contributions to ( ) per residue for homologous proteins as functions of the residue number using a consensus sequence, with insertions treated by summing the contribution of the entire insertion as one residue. The results for homologous proteins should be examined for differences in the contributions to ( ) per residue that correlate with observed redox potential differences. These differences can then be correlated with any other sequence-redox potential data for proteins that lack crystal or NMR structures. In addition, any sequences of homologous proteins that lack both redox potentials and structures should be examined, because residues important in defining the redox potential are likely to have semi-sequence conservation of a few key amino acid types. 

The elegant genetic studies by the group of Charles Yanofsky at Stanford University, conducted before the crystal structure was known, confirm this mechanism. The side chain of Ala 77, which is in the loop region of the helix-turn-helix motif, faces the cavity where tryptophan binds. When this side chain is replaced by the bulkier side chain of Val, the mutant repressor does not require tryptophan to be able to bind specifically to the operator DNA. The presence of a bulkier valine side chain at position 77 maintains the heads in an active conformation even in the absence of bound tryptophan. The crystal structure of this mutant repressor, in the absence of tryptophan, is basically the same as that of the wild-type repressor with tryptophan. This is an excellent example of how ligand-induced conformational changes can be mimicked by amino acid substitutions in the protein. 

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

Both enzymes belong to the family of a,p-hydrolases." The active site of MeHNL is located inside the protein and connected to the outside through a small channel, which is covered by the bulky amino acid tryptophane 128." It was possible to obtain the crystal structure of the complex with the natural substrate acetone cyanohydrin with the mutant SerSOAla of MeHNL. This complex allowed the determination of the mode of substrate binding in the active site." A summary of 3D structures of known HNLs was published recently." " ... 

Figure 18.4 Structures of heme/Cu oxidases at different levels of detail, (a) Position of the redox-active cofactors relative to the membrane of CcO (left, only two obligatory subunits are shown) and quinol oxidase (right), (b) Electron transfer paths in mammalian CcO. Note that the imidazoles that ligate six-coordinate heme a and the five-coordinate heme are linked by a single amino acid, which can serve as a wire for electron transfer from ferroheme a to ferriheme as. (c) The O2 reduction site of mammalian CcO the numbering of the residues corresponds to that in the crystal structure of bovine heart CcO. The subscript 3 in heme as and heme 03 signifies the heme that binds O2. The structures were generated using coordinates deposited in the Protein Data Bank, lari [Ostermeier et al., 1997] Ifft [Abramson et al., 2000] (a) and locc [Tsukihara et al., 1996] (b, c).

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