Function loop regions

The secondary structure elements, formed in this way and held together by the hydrophobic core, provide a rigid and stable framework. They exhibit relatively little flexibility with respect to each other, and they are the best-defined parts of protein structures determined by both x-ray and NMR techniques. Functional groups of the protein are attached to this framework, either directly by their side chains or, more frequently, in loop regions that connect sequentially adjacent secondary structure elements. We will now have a closer look at these structural elements. 

Long loop regions are often flexible and can frequently adopt several different conformations, making them "invisible" in x-ray structure determinations and undetermined in NMR studies. Such loops are frequently involved in the function of the protein and can switch from an "open" conformation, which allows access to the active site, to a "closed" conformation, which shields reactive groups in the active site from water. 

The simplest motif with a specific function consists of two a helices joined by a loop region. Two such motifs, each with its own characteristic geometry and amino acid sequence requirements, have been observed as parts of many protein structures (Figure 2.12). 

Figure 2.12 Two a helices that are connected by a short loop region in a specific geometric arrangement constitute a helix-turn-helix motif. Two such motifs are shown the DNA-binding motif (a), which is further discussed in Chapter 8, and the calcium-binding motif (b), which is present in many proteins whose function is regulated by calcium.

The loop region between the two a helices binds the calcium atom. Carboxyl side chains from Asp and Glu, main-chain C =0 and H2O form the ligands to the metal atom (see Figure 2.13b). Thus both the specific main-chain conformation of the loop and specific side chains are required to provide the function of this motif. The helix-loop-helix motif provides a scaffold that holds the calcium ligands in the proper position to bind and release calcium. 

Domains are formed by different combinations of secondary structure elements and motifs. The a helices and p strands of the motifs are adjacent to each other in the three-dimensional structure and connected by loop regions. Sequentially adjacent motifs, or motifs that are formed from consecutive regions of the primary structure of a polypeptide chain, are usually close together in the three-dimensional structure (Figure 2.20). Thus to a first approximation a polypeptide chain can be considered as a sequential arrangement of these simple motifs. The number of such combinations found in proteins is limited, and some combinations seem to be structurally favored. Thus similar domain structures frequently occur in different proteins with different functions and with completely different amino acid sequences. 

In almost every one of the more than 100 different known a/p structures 1 of this class the active site is at the carboxy edge of the p sheet. Functional residues are provided by the loop regions that connect the carboxy end of the strands with the amino end of the a helices. In this one respect a fun-I damental similarity therefore exists between the a/p-barrel structures and the I open a/p-sheet structures. 

The a/p-barrel structure is one of the largest and most regular of all domain structures, comprising about 250 amino acids. It has so far been found in more than 20 different proteins, with completely different amino acid sequences and different functions. They are all enzymes that are modeled on this common scaffold of eight parallel p strands surrounded by eight a helices. They all have their active sites in very similar positions, at the bottom of a funnel-shaped pocket created by the loops that connect the carboxy end of the p strands with the amino end of the a helices. The specific enzymatic activity is, in each case, determined by the lengths and amino acid sequences of these loop regions which do not contribute to the stability of the fold. 

The number of possible ways to form antiparallel p structures is very large. The number of topologies actually observed is small, and most p structures fall into these three major groups of barrel structures. The last two groups—the Greek key and jelly roll barrels—include proteins of quite diverse function, where functional variability is achieved by differences in the loop regions that connect the p strands that build up the common core region. 

Serpins form very tight complexes with their corresponding serine pro-teinases, thereby inhibiting the latter. A flexible loop region of the serpin binds to the active site of the proteinases. Upon release of the serpin from the complex its polypeptide chain is cleaved by the proteinase in the middle of this loop region and the molecule is subsequently degraded. In addition to the active and cleaved states of the serpins there is also a latent state with an intact polypeptide chain that is functionally inactive and does not bind to the proteinase. 

The functional reaction center contains two quinone molecules. One of these, Qb (Figure 12.15), is loosely bound and can be lost during purification. The reason for the difference in the strength of binding between Qa and Qb is unknown, but as we will see later, it probably reflects a functional asymmetry in the molecule as a whole. Qa is positioned between the Fe atom and one of the pheophytin molecules (Figure 12.15). The polar-head group is outside the membrane, bound to a loop region, whereas the hydrophobic tail is... 

The overall structure of the variable domain is very similar to that of the constant domain, hut there are nine p strands instead of seven. The two additional p strands are inserted into the loop region that connects p strands C and D (red in Figure 15.8). Functionally, this part of the polypeptide chain is important since it contains the hypervariahle region CDR2. The two extra p strands, called C and C", provide the framework that positions CDR2 close to the other two hypervariahle regions in the domain structure (Figure 15.8). 

We have sequenced RpII and studied the structures of RpII and RpIII in solution by 2D-NMR and distance geometry methods. The resonances are almost completely assigned, and secondary and tertiary structures have been determined. Our results indicate that Rp toxins have a four strand anti-parallel )9-sheet and no a-helix. Functionally important residues are found to be located in looped regions of the... 

Single-site amino acid alterations were carried out to show that mutations in the T1 domain loop region disrupt p subunit binding to the a subunit and abolish inactivation. Similar mutations on the p subunit surface that contacts the T1 domain also affected inactivation. The conclusion is that both subunits are necessary for inactivation peptides to carry out their function. 

More recently, the Pam amino acid chimera has also been incorporated into a small j0j0a-motif peptide scaffold [28]. The family of BBA peptides was developed in our laboratory as structured platforms for the design of functional motifs. These motifs are attractive because they are small enough (23 residues) to be easily synthesized by standard solid phase synthesis methods. Additionally, the motifs appear to possess sufficient structural complexity to influence coenzyme properties while still being amenable to structural characterization by standard spectroscopic techniques [3, 29, 30]. The BBA peptides include a -hairpin domain with a type IT turn connected by a loop region to an a-heli-cal domain (Fig. 10). Packing of the sheet and helix against one another is accomplished by hydrophobic contacts created by a hydrophobic core of residues. 

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