Protein structure inaccessible residues

As stated earlier, lipases act at the interface between hydrophobic and hydrophilic regions, a characteristic that distinguishes lipases from esterases. Similar to serine proteases, lipases share the nucleophile-histidine-acidic residue catalytic triad that manifests itself as either a Ser-His-Asp triad or a Ser-His-Glu triad. The enzyme s catalytic site often is buried within the protein structure, surrounded by relatively hydrophobic residues. An a-helical polypeptide structure acts as a cover, making the site inaccessible to solvents and substrates. For the lipase to be active, the a-helical lid structure has to open so that the active site is accessible to the substrate. The phenomenon of interfacial activation is often associated with reorientation of the lid, increasing the hydrophobicity of the surface in the vicinity of the active site and exposing it. The opening of the lid structure may be initiated on interaction with an oiFwater interface. 

Structure of the active center. The active centers of this dimeric enzyme are so well embedded into its protein structure that they are inaccessible to the solvent. The two centers are situated approximately 30 A apart from each other but connected by /3-strands. The active center consists of a type 2 copper center and a cofactor. Sequence comparisons have established that the residues His 8, His 246, and His 357 coordinate the copper ions in both yeast and plants (e.g., lentil seeds) [120,122]. The participating cofactor is typical for amine oxidases, diamine oxidases, and lysyl oxidases but has not yet been found in any other protein - 2,4,5-trihydroxy-phenylalanine quinone [123, 124] (also known as TOPA-quinone, TPQ or 6-hydroxy-DOPA quinone), an internal cofactor which is created by post-translational modification of the tyrosine in position 387 [120]. The consensus sequence of the amino acids neighboring the TOPA cofactor are conserved in all known amine oxidases - Asn-TOPA-Asp/Glu [113,120, 123,125-127]. The positions of the histidine ligands relative to TOPA quinone are conserved in all known amine oxidases as well. The chain lengths of the amine oxidase monomers vary according to the organism of origin 692 residues in yeast [128], 762 in bovine serum amine oxidase [128,129] and 569 in the enzyme from lentil seeds [120,130]. 

Detailed pictures of the iron-binding sites in transferrins have been provided by the crystal structures of lactoferrin (Anderson et ai, 1987, 1989 Baker etai, 1987) and serum transferrin (Bailey etal., 1988). Each structure is organized into two lobes of similar structure (the amino- and carboxy-terminal lobes) that exhibit internal sequence homology. Each lobe, in turn, is organized into two domains separated by a cleft (Fig. 3 and 10). The domains have similar folding patterns of the a//3 type. One iron site is present in each lobe, which occupies equivalent positions in the interdomain cleft. The same sets of residues serve as iron ligands to the two sites two tyrosines, one histidine, and one aspartate. Additional extra density completes the octahedral coordination of the iron and presumably corresponds to an anion and/or bound water. The iron sites are buried about 10 A below the protein surface and are inaccessible to solvent. 

Structural studies95-97,101 103 on cytochromes of the c and c2 types show that the heme group provides a core around which the peptide chain is wound. The 104 residues of mitochondrial cytochrome c are enough to do little more than envelope the heme. In both the oxidized and reduced forms of the protein, methionine 80 (to the left in Fig. 16-8A) and histidine 18 (to the right) fill the axial coordination positions of the iron. The heme is nearly "buried" and inaccessible to the surrounding solvent. 

Sequences of the representative proteins are displayed in JOY protein sequence/ structure representation (http //www-cryst.bioc.cam.ac.uk/ joy/). The representations are uppercase for solvent inaccessible, lowercase for solvent accessible, red for a helix, blue for strand, maroon for 310 helix, bold for hydrogen bond to main chain amide, underline for hydrogen bond to main chain carbonyl, cedilla for disulfide bond, and italic for positive angle. The query sequence is displayed in all capital letters. The consensus secondary structure (a for a helix, b for strand, and 3 for 310 helix) as defined, if greater than 70% of the residues in a given position in that particular conformation, is given underneath. 

Trypsin attacks the peptide bonds following the basic amino acids arginine and lysine. Formation of chloramines decrease trypsin binding sites, which causes a decrease in protein susceptibility to trypsin digestion. On the other hand, chloramine formation from free amino residues may induce changes in tertiary albumin structure, revealing some normally inaccessible amino residues. Therefore, removal... 

The lack of reversibility, and the presence of precipitation, makes thermodynamic analysis of aFGF particularly challenging. Precipitation in the presence of DTT indicates that precipitation is not dependent upon the formation of mixed disulfides. Structural analysis of human aFGF (17) shows that the three free cysteine residues are located at solvent inaccessible positions (figure 4). Thus, formation of mixed disulfides would be expected to destabilize the protein because a) structural changes would be required to expose the cysteines for oxidation and b) covalent adducts of the cysteine residues would have to be tolerated within the packing constraints of the interior of the protein for the native state to be adopted. 

The structure of the Src SH2 domain in complex with two low-affinity tyrosyl phosphopeptides elucidated the structural basis for pTyr recognition by SH2 domains (Waksman et al., 1992). The pTyr is stabilized by a dense network of hydrogen bonds and ionic interactions contributed by SH2 domain residues forming a deep cavity, the pTyr binding pocket (Fig. 2B). Most noteworthy, a universally conserved arginine residue (Arg PB5) at the center and base of the cavity makes a bidentate ionic interaction with two oxygens of the phosphate group. Arg pB5 is nearly completely solvent inaccessible in the free form of the protein. In the bound form, the ionic interaction which Arg pB5 makes with the phosphate is also entirely removed from solvent. 

Fig. 6. 24 A depiction of the X-ray structure of Ca-BFl. The right part of the protein is the kringle-domain, where the solvent inaccessible tryptophan residues Trp90 and Trpl26 are located. The Gla-domain is the left part of the protein, containing the solvent and quencher accessible Trp42 and seven calcium ions (dots).

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