Protein Data Preparation

Preparation of a Brookhaven Protein Data Bank (PDB)-formatted [10] file containing the coordinates and appropriate names of all atoms, including all polar and aromatic hydrogens. 

FIGURE 15.4 Computer record of a two-dimensional capillary electrophoresis analysis of a protein homogenate prepared from ahiopsy obtained from the fundus of a Barrett s esophagus patient. The data were generated hy performing 1 s transfers between capillaries and a 9 s second-dimension separation. The first-dimension separation employed the same buffer as the CSE separation in Fig. 15.1 and the second-dimension separation employed the same buffer as the MECC separation in Fig. 15.1. 

Structural biology provides a final way to define an E2 enzyme. As expected from the strong sequence conservation, the E2 core domain adopts a conserved fold. At the time this article was being prepared, twelve different E2 structures had been deposited in the Protein Data Bank. The average root-mean-square deviation of... 

I wish to thank Andrew B. Carrell, who assisted with many of the calculations and figure preparations necessary for the production of this chapter. I also thank H. L. Carrell, G. D. Markham, A. S. Mildvan, and E. K. Patterson for many helpful discussions. Ball-and-stick drawings were drawn with the computer program VIEW (Carrell, 1976). Use of the Cambridge Structural Database (Allen etal., 1979) and the Protein Data Bank (Bernstein el al., 1977) is acknowledged. This work was supported by National Institutes of Health grants GM 44360, CA 10925, and CA 06927 and by an appropriation from the Commonwealth of Pennsylvania. 

Other Protein Components. Other protein components In complex food systems and In protein Ingredient preparations may Interfere with or modify gelation reactions. Protein Interaction between whey protein and casein upon heating has a profound Influence on the characteristics of the casein gel structure In cheesemaking. Similarly protein Interactions are Important to meat structures. Protein-protein Interaction between soy and meat proteins has also been demonstrated with heat treatment (28). While concrete Interaction data have not been collected on protein gels formed from protein combinations, gelation properties of whey proteln/peanut flour blends have been Investigated GU) ... 

Plate 1 Stereo photograph of Zif268/DNA complex (see N. P. Pavletich and and C. O. Pabo, Science 252, 809, 1991). (For discussion, see Chapter l. )NOTE Atomic coordinates for preparing this display were obtained from the Protein Data Bank (PDB), which is described in Chapter 7. The PDB file code is lzaa. To expedite your access to all models shown in this book, I provide file codes in this format PDB lzaa. Image created by Swiss-PdbViewer, rendered by POV-Ray. To obtain these programs, see the CMCC Reader s Page. 

The tubers of Jerusalem artichoke typically comprise about 80% water, 15% carbohydrate, and 1 to 2% protein. Data on the composition of Jerusalem artichoke are relatively sparse in comparison to other vegetables, however, and significant variation has been recorded for certain parameters. Differences in cultivar, time of harvest, production conditions, postharvest treatment, and preparation methods most likely account for this variation (Table 5.1). 

With these newer methods of protein separation and amino acid analysis he prepared serum protein fractions by serial salting out with ammonium sulfate and by the Sober and Peterson DEAE cellulose columns (42), using the sera of reptile, fowl, and mammalian blood. Some of the amino acid analyses were carried out by the automatic amino acid methods of Hirs, Moore, and Stein (18). Fortified with this plethora of data, Block now had the opportunity to re-examine not only the ratio of the basic amino acids, but at least 12 amino acids in a variety of protein fractions prepared by at least two different procedures. With the aid of a statistician he determined the significance of the constancy of the molar ratios of pairs of amino acids and found that in spite of the marked variation of the absolute amounts of an amino acid, the molar ratios of certain pairs remain relatively constant among the numerous protein components of animal sera. 

Fig. 3. Artificial substrates for the insulin receptor tyrosyl kinase. Shows the Km values exhibited by basal and insulin-stimulated kinase activities together with the insulin-stimulated increase in Vmax for a variety of substrates. These include angiotensin and its modified derivative (VAL-5), also synthetic peptides of Glu Tyr and the so-called sarc -peptide, which bears the sequence around the tyrosyl autophosphorylation site of the sarc protein. Data are also given for a G-protein mixture of Gj/G0. These studies (referred to in the text) all employed soluble, purified insulin receptor preparations. No evidence has yet been presented for tyrosyl phosphorylation of substrates using isolated membrane preparations containing insulin receptors.

Figure 2. Dependence of unfolding rate constants on pH. Wild type tailspike protein was prepared in 50 mM Tris, 1.7 mM 2-mercaptoethanol and 2% SDS and adjust to different pH values by 1 N HCl. Thermal unfolding was done at 65°C and followed by SDS-PAGE at about 20°C. Sample pH values shown here have been corrected to 65°C. kj (a) and k2 ( ) shown in log are the thermal unfolding rate constants for the conversions from N to I and from I to M, respectively. The linear lines through the data points are the results of least-square fit to each individual pH phase for both kj and k2 data. The calculated slopes of the fitting lines for kj are -0.46 and 0.35 for the low and high pH phases, respectively and for k2 are -1.9 and 1.1 for the low and high pH phases, respectively.

Using the reference protein to prepare calibrators resulted in an approximate threefold overestimation for samples prepared from the lyophilized drug. As would be expected, a similar degree of underestimation was seen when calibrators were prepared from the lyophilized material and used to test liquid solutions of reference material. Clearly, this example illustrates the fact that formulation or other manufacturing changes can affect relative immunoreactivity of protein biotherapeutics, presenting challenges for accurate quantitation. One would be advised to match materials for calibration with the test samples whenever possible, and certainly when insufficient characterization data exist to assure that materials are equivalent. 

Fig. 1. Ribbon representation of an afiy unit of the crystallographic trimer of K. aerogeties urease. The Ni ions are located at the top of the a subunit (light gray) and are accessible from the solvent medium. The / and y subunits are depicted in dark and medium grey respectively. This drawing was prepared with Molscript [126] by A. Volbeda. Coordinates were obtained from entry 1KAU [16] from the Protein Data Bank [127]...

Figure 7.9 Data-dependent microfluidic LC-MS-MS analysis of a protein fraction prepared from the MCF7 breast cancer cell line (SCX fraction eluted with 50-70 mM NaCl). Conditions 2 cm long x 50 pm deep channel, Zorbax SB-( I S c/, — 5 pm packing material, 2 x 200 pumping channels ( 1.5 pm deep), 2 x 100 valving channels, LC eluent NH4HCO3 (15mM) in H20 CH3OH (40 60) at pH 8, flow rate 60-70 nLmin-1. (Reprinted with permission from ref. 33).

Figure 3.10 (a) A stereo drawing of the Hb tetramer, extracted from the Protein Data Bank based on the work of Perutz and coworkers (Fermi et al 1984). The dots are ordered water molecules. Drawing prepared by Dr J. Rafteiy. (b) A Beevers molecular model of the tetramer (with permission to be reproduced here). 

FIGURE 9.61 Molecular structures of a 1,4-p-endoglucanase (a) and 1,4-p-exoglucanase (b) showing the cleft and tunnel features, respectively. These drawings were prepared from files downloaded from the Brookhaven Protein Data Bank (http //pdb.pdb.bnl.gov). (a) (ICEC) and (b) (3CBH, Ca skeleton) were rotated to reveal the cleft and tunnel, respectively, with RasMol, Version 2.5, by Roger Sayle, freeware available from the Brookhaven National Laboratory web site. 

B A space-filling view of all water molecules detected by X-ray diffraction demonstrates the internal waters of ITiales available to function by the consilient mechanism for protein-based machines. (Figure preparation was based on the crystallographic data of Abrahams et al. obtained from the Protein Data Bank, Structure File IBMF.)... 

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