Folded protein flexibility

Although the underlying physics and mathematics used to convert relaxation rates into molecular motions are rather complex (Lipari and Szabo, 1982), the most important parameter obtained from such analyses, the order parameter. S 2, has a simple interpretation. In approximate terms, it corresponds to the fraction of motion experienced by a bond vector that arises from slow rotation as a rigid body of roughly the size of the macromolecule. Thus, in the interior of folded proteins, S2 for Hn bonds is always close to 1.0. In very flexible loops, on the other hand, it may drop as low as 0.6 because subnanosecond motions partially randomize the bond vector before it rotates as a rigid body. 

Because of the intrinsic flexibility and poor resonance dispersion of unfolded and partly folded proteins, long-range NOEs are generally very difficult to observe and assign. While observation of a long-range NOE between two protons provides a definitive indication that they are in close proximity in at least some structures in the conformational ensemble, determination of the nature of the folded structures is difficult unless an extensive network of NOEs can be observed. This has so far been achieved in only one case (Mok et al., 1999). 

Fig. 4. Schematic representation of template-assembled synthetic proteins. The conforma-tionally restricted template can be orthogonally protected and sequentially linked to helical segments to form a large variety of functionalized TASP proteins. Flexible spacers that connect the folded peptide segments and the template provide the necessary conformational freedom that will allow the hydrophobic residues to find their optimum orientations for packing the core...

Further experiments gave more clues. There are enzymes (called proteases) that have the ability to chew up other proteins, decomposing them into amino acids. When a small amount of a protease is added for a short time to a solution containing cilia, the protease quickly slices up the nexin linkers at the edge of the structure. The rest of the cilium remains intact. The reason that the protease rapidly attacks the linkers is that, unlike the other proteins of the cilium, the nexin linkers are not folded up tightly instead, they are loose, flexible chains. Because they are loose, the protease can cut them as rapidly as a pair of scissors can cut a paper ribbon. (The protease cuts tightly folded proteins as rapidly as scissors cut a closed paperback book.)... 

The lessons we have learned from physics are of a different nature. The history of physics is replete with examples of the elucidation of connections between what seem to be distinct phenomena and the development of a unifying framework, which, in turn, leads to new observable consequences [13]. Indeed, strong evidence suggests that globular proteins share many common characteristics their ability to fold rapidly and reproducibly in order to create a hydrophobic core, the fact that there seem to be a relatively small number (on the order of a few thousand) of distinct modular folds made up of helices and almost planar sheets, the fact that protein folds are flexible and versatile in order to accomplish the dizzying array of functionalities that these proteins perform, and the unfortunate tendency of proteins to aggregate and form amyloids, which are implicated in human diseases. 

It is assumed that the limited proteolysis phenomenon derives from the fact that a specific polypeptide chain segment of the compact, folded protein substrate is exposed and flexible so that it can fit the active site of the appropriate peptidase for an efficient and selective limited hydrolysis. There is no doubt that enhanced chain flexibility or segment mobility is the key feature of the site of peptide bond hydrolysis demonstrated by a clear-cut correlation between sites of proteolytic attack and sites of enhanced chain flexibility. The present availability of automatic, efficient and sensitive techniques of protein sequencing and, particularly, the recent dramatic advances of mass spectrometry 361 in the analysis of peptides and proteins, allows a more systematic use of the limited proteolysis approach as a simple first step in the elucidation of structure-dynamics-function relationships for novel proteins which are only available in minute amounts. 

Detailed models of the folded states of proteins depend almost entirely on X-ray diffraction analysis of the protein crystal. Although side chains and flexible loops on the surface may be mobile in solution, protein conformation in solution is essentially that determined in the solid crystal. The atoms of folded proteins are generally well fixed in space. 

The result of the two types of folding of the polypeptide main chain—the a helix and the p sheet—is that the architect is provided with two different construction modules which can be joined together (by loops), given very stable entities that are often quite rigid. The loops serve as the main foci for flexibility and variability. The a helix, the p sheet and loops are the three components that, with slight variations (such as the 3 o helix), will be used in the final conformation of the folded protein. 

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