Polypeptides structural mapping

A comparison of the maps of Figs. 15 and 16 illustrates similar additional steric restrictions on a poly-L-alanine chain, as one passes from a dipeptide (Fig. 15) to helical structures (Fig. 16). Further discussion of steric effects in small polypeptide structures can be found in the recent review of Ramachandran and Sasisekharan (1968). 

L. Structural Mapping of Polypeptides and Proteins on Reversed Phases... 

Hearn, M. T. W., 1980, The use of reverse-phase high-performance liquid chromatography for the structural mapping of polypeptides, / Liquid Chromatogr. 3 1255-1276. 

Early controlled proteolysis experiments (i, 4) clearly showed that the CAD polypeptide was organized into discrete functional domains. The complete domain structure of the polypeptide was mapped using three approaches. Proteolytic fragments were isolated, partially sequenced and their function was determined (6-8). The CAD cDNA (5, 9-12) was sequenced and the identity of the domains and the domain junctions were determined by comparison with the sequence of monofunctional bacterial proteins. More recently, many of the domains and subdomains were cloned and characterized (13-21). The... 

Figure 8.3 The DNA-binding protein Cro from bacteriophage lambda contains 66 amino acid residues that fold into three a helices and three P strands, (a) A plot of the Ca positions of the first 62 residues of the polypeptide chain. The four C-terminal residues are not visible in the electron density map. (b) A schematic diagram of the subunit structure. a helices 2 and 3 that form the helix-turn-helix motif ate colored blue and red, respectively. The view is different from that in (a), [(a) Adapted from W.F. Anderson et al., Nature 290 754-758, 1981. (b) Adapted from D. Ohlendorf et al., /. Mol. Biol. 169 757-769, 1983.]...

From a map at low resolution (5 A or higher) one can obtain the shape of the molecule and sometimes identify a-helical regions as rods of electron density. At medium resolution (around 3 A) it is usually possible to trace the path of the polypeptide chain and to fit a known amino acid sequence into the map. At this resolution it should be possible to distinguish the density of an alanine side chain from that of a leucine, whereas at 4 A resolution there is little side chain detail. Gross features of functionally important aspects of a structure usually can be deduced at 3 A resolution, including the identification of active-site residues. At 2 A resolution details are sufficiently well resolved in the map to decide between a leucine and an isoleucine side chain, and at 1 A resolution one sees atoms as discrete balls of density. However, the structures of only a few small proteins have been determined to such high resolution. 

In early 1990 it became apparent that the structure of galactose oxidase from Dactylium dendroides was about to emerge. A 2.5 A multiple isomor-phous replacement (MIR) map based on area detector data from a native and three derivative crystals yielded a polypeptide chain tracing. The refined structure at 1.9 A (R = 0.179) (Ito et al., 1991) shows that galactose oxidase consists of three domains, each of which is predominandy jS... 

Nuclease behaves like a typical globular protein in aqueous solution when examined by classic hydrodynamic methods (40) or by measurements of rotational relaxation times for the dimethylaminonaphth-alene sulfonyl derivative (48)- Its intrinsic viscosity, approximately 0.025 dl/g is also consistent with such a conformation. Measurements of its optical rotatory properties, either by estimation of the Moffitt parameter b , or the mean residue rotation at 233 nin, indicate that approximately 15-18% of the polypeptide backbone is in the -helical conformation (47, 48). A similar value is calculated from circular dichroism measurements (48). These estimations agree very closely with the amount of helix actually observed in the electron density map of nuclease, which is discussed in Chapter 7 by Cotton and Hazen, this volume, and Arnone et al. (49). One can state with some assurance, therefore, that the structure of the average molecule of nuclease in neutral, aqueous solution is at least grossly similar to that in the crystalline state. As will be discussed below, this similarity extends to the unique sensitivity to tryptic digestion of a region of the sequence in the presence of ligands (47, 48), which can easily be seen in the solid state as a rather anomalous protrusion from the body of the molecule (19, 49). 

The 13C chemical shift contour map for the Cp carbon of the L-alanine residue in peptides and polypeptides was made as a function of the dihedral angles(, W) by using the experimental data. Also, the corresponding calculated map was made by using the ab initio coupled Hartree-Fock method with the gauge included atomic orbitals(GIAO-CHF). From these results, it was found that the calculated map explains the chemical shift behavior of the a-helix and p-sheet forms in poly(L-alanine) and some proteins. This suggests that the calculated map is applicable to the structural analysis of proteins with complicated structure. 

So far, we have investigated higher-order structure of polypeptides by solid-state high-resolution NMR not only using experimental but also theoretical methods[2-4]. The chem cal shifts can be characterized by variations in the electronic states of the local conformation as defined by the dihedral angles(4>,W). Ando et al. have calculated contour map for the Cp carbons of an alanine dipeptide by using the FPT INDO method within the semi-empirical MO framework. The calculated map reasonably predicts the experimental version. This shows that the chemical shift behavior of the L-alanine residue Cp-carbonyl carbons in the... 

In vacuo most peptides are constrained to quasi-planar conformations (, i/i 0°, 180°), while Polarizable Continuum Model (PCM) calculations show that in aqueous solution another stable structure appears for 4> -60°, tft -60° this is noteworthy because such angles are typical of a-helix conformations of polypeptides, which is particularly favoured by the solvent [2], This feature is illustrated in Figure 3.2, where Ramachandran maps (i.e. plots of the energy versus 4> and tft) are reported both in vacuo and in aqueous solution. 

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