Native protein structures approximation

FIGURE 4-15 Globul ar protein structures are compact and varied. Human serum albumin (Mr 64,500) has 585 residues in a single chain. Given here are the approximate dimensions its single polypeptide chain would have if it occurred entirely in extended /3 conformation or as an a helix. Also shown is the size of the protein in its native globular form, as determined by X-ray crystallography the polypeptide chain must be very compactly folded to fit into these dimensions. 

One can dso obtain NMR spectra for proteins in micelles, which may allow the study of membrane protein structure in an environment approximating their native one. A combination of labels (l N,13c) was used for NMR studies of detergent-solubilized M13 coat protein. Although most of die resonances in the spectrum have not been assigned, there was clear indication that many of the protein residues had two distinct resonances of equal intensity. This was interpreted to mean (in combination with the results of sedimentation equilibrium, Raman and CD studies) that the protein was present in two conformers that represent the non-equivalent monomers of an asymmetric dimer. NMR has also been used to determine the spatial structures of gramicidin A and -labeled bacteriorhodopsin fragments in a membrane-like milieu b... 

Likewise, the strucmre of subtilisin (pH 3.0) suspended in varying ratios of acetonitrile and water demonstrated a-helical content similar to that in the lyophilized powder (Griebenow and Klibanov, 1996). Furthermore, the rate of transesteriflcation reactions of subtilisin (pH 7.8) suspended in DMSO/acetonitrile, formamide/acetonitrile or formamide/dioxane were increased approximately 100-fold over aqueous conditions (Almarsson and Klibanov, 1996). Similar results were obtained for subtilisin (pH 7.8) in a tetrahydrofuran/1-propanol mixture (Affleck et al., 1992). These results can be attributed to the increased structural rigidity of the active conformation of the protein in the solid, and the denaturing characteristics of the solvent at the solvent-particulate interface. Preservation of this molecular memory or molecular imprint of the protein can also be used to stabilize structure and activity (Mishra et al., 1996 Rich and Dordick, 1997 Santos et al., 2001). Subtilisin was lyophilized from crown ethers, resulting in more native like structure, by FTIR, and increased enzyme activity in THF, acetonitrile and dioxane (Santos et al.,2001). 

I Q) of the unlabelled protein is denoted as In and that for the labelled protein is /jj +122 + /n, where I22 is the scattering from the markers and 1 2 is the cross-term. The experimental procedure is based on the subtraction of the curve for the native protein from that of the labelled protein to give an oscillatory curve whose periodicity gives the required distance between metal sites. The subtraction assumes A2 to be negligible test calculations show that there must be > 200 electrons in each marker for a protein of 100,000 for this to be a satisfactory approximation. Thus the distance between the tetra-mercury markers attached to Cys 93 in the two chains of haemoglobin was determined as 3.8 + 0.2 nm, in good accordance with a calculation of 3.76 nm from the crystal structure [162]. The distance between mercury labels on histidine decarboxylase was determined as 6.9 0.3 nm [162]. 

The slow rate is usually small enough that the contribution from structured amides is negligible, thus a peptide that is 50% a-helix and 50% random coil in the native protein should become approximately 50% exchanged and no more over the first few seconds. This makes it possible to test the extent to which a crystal structure reflects native structural ensemble of a protein in solution [6, 21] if the observed level of exchange in a particular peptide is substantially above that expected from the crystal structure, then the protein is less structured than reported. Alternatively, if the observed level of exchange is substantially lower than that expected from the crystal structure, then the protein is more structured than reported. 

Two additional decoy data sets of misfolded proteins [17] and of predicted protein structures from the Critical Assessment of Techniques for Protein Stmcture Prediction (CASP) [67] are also used to illustrate the method and its utility. Individual components of the energy perform worse than the total energy for example, for the bulk of the well-packed decoys, the van der Waals energy provides very little information about structural similarity between a well-packed non-native structure and the native state. It is also shown that some aspects of the SGB model results can be mimicked by a screened electrostatic energy, although the SGB approximation provides a better discriminatory measure between non-native and native states. 

Ubiquitin has been the subject of a number of experimental studies as it is a small and soluble protein without disulfide linkages. The protein has a well-defined and packed hydrophobic core. There are a number of kinetic folding studies on this system, in addition to a high resolution crystal structure. 2D NMR studies of a partially unfolded form of the protein in 60% methanol indicate that approximately one-half of the molecule contains dismpted but native-like structure, while the other half is unstmctured and/or contains non-native structure. The interpretation of hydrogen-exchange experiments have led to the conclusion that this state is native-like. ... 

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