Multidomain proteins

Peptidases have been classified by the MEROPS system since 1993 [2], which has been available viatheMEROPS database since 1996 [3]. The classification is based on sequence and structural similarities. Because peptidases are often multidomain proteins, only the domain directly involved in catalysis, and which beais the active site residues, is used in comparisons. This domain is known as the peptidase unit. Peptidases with statistically significant peptidase unit sequence similarities are included in the same family. To date 186 families of peptidase have been detected. Examples from 86 of these families are known in humans. A family is named from a letter representing the catalytic type ( A for aspartic, G for glutamic, M for metallo, C for cysteine, S for serine and T for threonine) plus a number. Examples of family names are shown in Table 1. There are 53 families of metallopeptidases (24 in human), 14 of aspartic peptidases (three of which are found in human), 62 of cysteine peptidases (19 in human), 42 of serine peptidases (17 in human), four of threonine peptidases (three in human), one of ghitamicpeptidases and nine families for which the catalytic type is unknown (one in human). It should be noted that within a family not all of the members will be peptidases. Usually non-peptidase homologues are a minority and can be easily detected because not all of the active site residues are conserved. 

Coacervation occurs in tropoelastin solutions and is a precursor event in the assembly of elastin nanofibrils [42]. This phenomenon is thought to be mainly due to the interaction between hydro-phobic domains of tropoelastin. In scanning electron microscopy (SEM) picmres, nanofibril stmc-tures are visible in coacervate solutions of elastin-based peptides [37,43]. Indeed, Wright et al. [44] describe the self-association characteristics of multidomain proteins containing near-identical peptide repeat motifs. They suggest that this form of self-assembly occurs via specific intermolecular association, based on the repetition of identical or near-identical amino acid sequences. This specificity is consistent with the principle that ordered molecular assembhes are usually more stable than disordered ones, and with the idea that native-like interactions may be generally more favorable than nonnative ones in protein aggregates. 

The previous ELP fusions all are examples of protein purification in which the ELP is covalently connected to the protein of choice. This approach is suitable for the purification of recombinant proteins that are expressed to high levels, but at very low concentrations of ELP the recovery becomes limited. Therefore this approach is not applicable for proteins expressed at micrograms per liter of bacterial culture, such as toxic proteins and complex multidomain proteins. An adjusted variant of ITC was designed to solve this problem. This variant makes use of coaggregation of free ELPs with ELP fusion proteins. In this coaggregation process, an excess of free ELP is added to a cell lysate to induce the phase transition at low concentrations of... 

It is equally important to work with full-length versions of enzymes whenever this is feasible. Some enzymes are expressed naturally as multidomain proteins in which the catalytic machinery is localized to a single, discrete protein domain. In... 

A major problem in unfolding studies of large proteins is irreversibility. In a study of elastase temperature-induced denaturation, second-derivative FTIR show a distinct loss of several sharp amide V features (dominant /3-sheet components and growth in broadened bands at 1645 and 1668 cm-1 (Byler et al., 2000). These features persisted on cooling, indicating lack of reversibility, a feature common to longer multidomain proteins. A graphic example of this is seen in the triosephosphate... 

Kolb, V. A., Makeyev, E. V., and Spirin, A. S. (2000). Co-translational folding of a eukaryotic multidomain protein in a prokaryotic translation system. J. Biol. Chem. 275, 16597-16601. 

Usually, TS /1-solenoids represent only parts of larger multidomain proteins. Other trimeric motifs found in these proteins include a-helical coiled coils, TS /1-spirals, trimeric bundles of single-stranded /1-solenoids, and irregular globular structures. Some of these domains may be needed for correct folding of the TS /1-solenoid. 

Several /i-solenoid domains appear to promote the oligomerization of multidomain proteins. There are at least three types of /i-solenoid association. First, oligomers (dimers or trimers) are formed by lateral interaction of the solenoids. For example, the C-terminal domain of the bacterial cell division inhibitor MinC is a short right-handed T-type solenoid with an apolar lateral face that mediates homodimerization (Cordell et al., 2001). Trimers of several bacterial transferases are formed by lateral, in-register, interaction of left-handed T-type /1-solenoids (Fig. 5). Second, dimers may form via interactions of the open terminal coils of /1-solenoids as in the dimeric structure of iron transporter stabilizer SufD (Badger et al., 2005). Finally, dimerization may be mediated by swapping of /1-strands of the terminal coils, as in the CAP (Dodatko et al., 2004) (Fig. S). 

The dependence of the residual dipolar coupling on the angle that the vector forms with a reference axis explains why the use of dipolar couplings makes possible the determination of the relative orientation of different domains in a multidomain protein and facilitates nucleic acid structure determination. Dipolar couplings can constitute up to 50% of the total structural data available for nucleic acids, while this number drops to 10-15% in proteins. Thus, the impact of the use of dipolar couplings on the structure determination of nucleic acids is generally more substantial than in the case of proteins. Furthermore, the presence or absence of tertiary structure in a protein or nucleic acid does not have a major influence on the number of dipolar couplings that can be measured, in contrast to the case of the NOE. 

In a multidomain protein whose domains have fixed orientations relative to each other, a unique alignment tensor will represent the preferred orientation of all the domains in the anisotropic environment. Therefore, structure refinement with dipolar couplings is performed as in one-domain proteins (Sect. 8.4). Several examples are reported in the literature of cases with conformational ambiguity due to the lack of NOE contacts between the domains. One example is the determination of subdomain orientation of the riboso-mal protein S4 z)41 [97]. In this work the lack of NOE contacts between the domains produces an ambiguity in interdomain orientation. The authors use two different anisotropic media to obtain dipolar couplings (DMPC/DHPC bicelles and Pfl filamentous bacteriophages). They conclude that subdomain orientation in solution is similar to the one present in the crystal structure. 

Fig. 2. An example of a complex multidomain protein that includes both domain concatenation and intercalation. (A) See color insert. RASMOL view of phosphotransferase pyruvate kinase (pdb entry lpkn) colored to show the three identifiable domains. Blue is the j3 barrel regulatory domain, orange is an eightfold a/fi barrel, the catalytic substrate binding domain, and green is a central /3, a/(B nucleotide binding domain. Not displayed is the leader subsequence composed of a random coil and short helix. (B) Linear order along the sequence of these components.

The unfolding behavior of multidomain proteins in the presence of preservatives has also been evaluated. For example, IL-1R (type I) has three domains that correspond to three unfolding transitions as measured by microcalorimetry and depicted in Figure 13.1.31 All three transitions exhibit some shift to lower Tm in the presence of the three preservatives tested (i.e., 0.065% phenol, 0.1% metacresol, and 0.9% benzyl alcohol), in comparison to a control containing no preservative. Such a destabilization could have consequences for the shelf-life of the product. Another example of the impact of preservatives on a multidomain protein is illustrated in Figure 13.5. The protein is fused to a single IgCi, Fc. The... 

Limited proteolysis. Flexible regions of proteins can sometimes be removed by digestion of the protein with different proteases. This technique is based on the techniques that were used to determine the core folded regions of proteins, most notably antibodies (Porter, 1973). Limited proteolysis can be used to remove flexible loops of proteins, or separate multidomain proteins into separate domains and has been used successfully in a number of instances (Noel et al., 1993 Sondek et al., 1996 Mazza et al., 2002). 

Other Class A polymerases. The Thermus aquati-cus (Taq) polymerase is best known for its widespread use in the polymerase chain reaction (PCR Fig. 5-47). Like E. coli I the enzyme is a large multidomain protein. The structure of the catalytic domains of the two enzymes are nearly identical, but the Taq polymerase has poor 3 -5 editing activity.276 The enzyme has been carefully engineered to improve its characteristics for use in the PCR reaction.277... 

A related gene family is present in the same gene cluster and encodes for proteins containing an S 100-like domain fused to a larger peptide. These proteins include trichohyalin, filaggrin, and repetin, which are multidomain proteins involved in epidermal differentiation (Marenholz et al., 2001 Huber et al., 2005) and are classified as a separate family. 

Krasnoperov VG, Beavis R, Chepumy OG et al (1996) The calcium-independent receptor of a-latrotoxin is not a neurexin. Biochem Biophys Res Commun 227 868-75 Krasnoperov VG, Bittner MA, Beavis R et al (1997) a-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor. Neuron 18 925-37 Krasnoperov VG, Bittner MA, Mo W et al (2002b) Protein tyrosine phosphatase-G is a novel member of the functional family of a-latrotoxin receptors. J Biol Chem 277 35887-95 Kreienkamp HJ, Zitzer H, Gundelfinger ED et al (2000) The calcium-independent receptor for a-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins. J Biol Chem 275 32387-90 Lajus S, Lang J (2006) Splice variant 3, but not 2 of receptor protein-tyrosine phosphatase a can mediate stimulation of insulin-secretion by a-latrotoxin. J Cell Biochem 98 1552-9 Lajus S, Vacher P, Huber D et al (2006) a-Latrotoxin induces exocytosis by inhibition of voltage-dependent K+ channels and by stimulation of L-type Ca2+ channels via latrophilin in [5-cells. J Biol Chem 281 5522-31... 

As most NRPS multienzymes are multidomain proteins with multiple activation domains, multiple sites may participate in the reactions assayed, and no clear result concerning a single specific site may result. In ACV synthetases, the nonadditivity of the initial rates has been observed in the S. clavuligerus enzyme [35] and the A. chrysogenum enzyme [1]. Two or more site activations of one substrate amino acid could be expected to depend on different binding constants, and thus be detectable by kinetic analysis. So far, however, no evidence for mixed types of concentration dependence has been found. It is thus not yet clear if nonadditivity results from misactivation or alteration of kinetic properties in the presence of multiple substrates. In the case of gramicidin S synthetase 2, evidence for misactivations has been reported [59],... 

These results demonstrate that, in analogy to the multidomain proteins in biosili-cification, triblock copolymers can direct the assembly of silica into complex structures. Furthermore, combining complex ABC copolymer architectures with the physical, electrical, and optical properties of inorganic materials holds considerable... 

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