Structural studies of proteins

Because the denatured state has long been thought to be essentially random and because of the inherent difficulties in defining and interpreting structural data averaged in very complex ways, protein chemists have been slow to take up the structural characterization of denatured proteins. Yet a more complete description of the relatively small amounts of persistent structure they display is not simply of academic interest. There are compelling reasons to pursue structural studies of proteins that are not folded. 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

Steitz, T. A., Structural studies of protein-nucleic acid interaction The sources of sequence-specific binding. Quar. Rev. 

Kainosho M, Tsuji T, Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution, Biochemistry, 21 6273-6279, 1982. 

In future high-resolution crystal structure studies of proteins, hydrogen bonding has to be analyzed in terms of two-center and three-center interactions. It should be stressed that three-center bonds may be especially important in protein dynamics because they can be regarded as transition intermediate from one two-center bond to another (see Part IV, Chap. 25). 

Although chronologically, structural studies of proteins in strongly protic solvents preceded those in weakly protic ones, it is advantageous to discuss the latter studies first. 

Table 2.4 Structural studies of proteins immobilized onto carbon nanotubes.

In structural studies of proteins/metalloproteins, concentrations of about 1 mM are typically required and proteins must be soluble and stable over a period of time (weeks). For small proteins with several tens of amino acids, e.g. metallothionine [27, 28], it is sufficient to use N-labeled samples to determine structures of the proteins. However, if proteins can be overexpressed in a bacterial system (e.g., Escherichia coli), it is desirable to overexpress the protein with uniform enrichment... 

Steitz, T.A. (1993) Structural Studies of Protein-Nucleic Acid Interaction The Sources of Sequence-Specific Binding, Cambridge University Press, Cambridge, UK. 

Solid films have been examined frequently for infrared analysis in biochemical work, for example, in structural studies of proteins, polypeptides, and polysaccharides. Such films have been of particular value for studying polarization spectra of macromolecules in intact films and in oriented ones (stretched, rolled, or stroked), thereby permitting knowledge to be gained concerning spatial arrangements within the molecule and conformational effects among molecules. (See The Use of Polarized Infrared Radiation and the Measurement of Dichroism, p. 73, for a detailed discussion.) A few workers have discussed the film technique (Lecomte, 1948 Randall et al., 1949 Hacskaylo, 1954). 

Fig. 1 The number of fold families identified from structural studies of proteins based on data obtained from SCOP. Representative structures are shown from each of the classes of protein fold, e.g., alpha or beta, that include enzymes. The proteins are represented as ribbons, colored from blue at the N-terminus through to red at the C-terminus. Cofactors and inhibitors are colored according to atom type and are represented in stick. For example, the alpha fold of Heme oxygenase is fold 143 out of 151 in the classification, a multihelical bundle containing two structural repeats of three-helical motif. The enzyme is an oxidoreductase EC 1.14.99.3 and is represented by the crystal structure of rat heme oxygenase-1 (HO-1) protein data bank acession code IDVG. (View this art in color at WWW. dekker. com.)...

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