Polypeptides analysis

Kangas, H., I. Ulmanen, T. Paunio, D.J. Kwiatkowski, M. Lehtovirta, A. Jalanko, and L. Peltonen. 1999. Functional consequences of amyloidosis mutation for gelsolin polypeptide — analysis of gelsolin-actin interaction and gelsolin processing in gelsolin knock-out fibroblasts. FEBS Lett. 454 233—9. 

Kitamoto N, Tanimoto S, Hiroi K, et al. Monoclonal antibodies to cowpox virus Polypeptide analysis of several major antigens. J Gen Virol. 1987 68 239-246. 

Reversed phase high performance liquid chromatography (RP-HPLC) now plays a critical role in the analysis and purification of peptides and proteins from natural and synthetic sources. The extraordinary popularity of RP-HPLC for polypeptide analysis can be attributed to a number of factors such as ... 

The microsecond phases have been studied at various pH. since It is know that this parameter influences the rate of electron transfer from Z to P-OOO" " (11.16.17). At the five pH values studied, the decay of the flash-induced absorption change at 820 nm can be fitted by three exponential components, with approximate (pH-Independant) half-times of 5 1 fjs, 25 3 /is, and over 1 ms. The middle phase makes up about 30 of the total, at ail pH values, but the proportion of fast phase varies from 13 at pH 5 to 52 at pH 9 (Fig. 1). The pH has no effect on the decay kinetics of the absorption changes in the absence of DBMIB. Polypeptides analysis of the (D1.D2) complex (1), together with results obtained by... 

For separation of thylakoid membrane components, thylakoid membranes of the cyanobacterium Synechococcus elongatus were solubilised by Triton X-100 (mass ratio Tritonichl = 25, final concentration of Triton 17.,v/v) or by SDS (mass ratio SDS chl = 50, final concentration of SDS 2.57.,v/v). For details see (5). E1ectrophoresis on gradient polyacrylamide gel (5-107., in case of pigment-proteins separation, 10-207. in case of polypeptide analysis) was performed in the buffer system described earlier (5,8). 

Polypeptide analysis of one of the PS-I mutants (Acfl04) showed a very good correlation in the quantity of CPI, 68kDa and other PS-I polypeptides, P-700 and the rate of PS-I electron transport. hcfXOA has lost 60% of all of these PS-I components and demonstrated the same 60% loss of the PS-I election transport... 

Translation of the atpB mRNA in the reticulocyte extract showed synthesis of polypeptides with a maximum Mr of about 52 kDa. Translation was neither stimulated nor inhibited in the presence of isolated photosynthetic membranes. atpB mRNA directed synthesis of a doublet polypeptide. Analysis of recovered, washed membranes showed that the beta subunit was associated with thylakoids (Fig 1). 

Nordlund I, Powlowski J, Shingler V. 1990. Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. Strain... 

FIGURE 4. Polypeptide analysis of 1, Prochloron thylakoids 2, spinach thylakoids 3, Prochloron Chi a- 4, spinach LHCP3 5, Prochloron Chi a - 6, CPI 7, Prochloron thylakoids. Lanes 1 and 2, samples boiled with SDS lanes 3-7, samples unboiled. Gels were silver stained. 

FIGURE 5. Polypeptide analysis of Chl-proteins isolated on a sucrose gradient. 1, spinach thylakoids 2, Prochloron Chi -b and Chi a-b 3, Prochloron CPI 4, protein standards. Samples were boiled with SDS and Coomassie Blue stained. 

Polypeptide analysis of Prochloron thylakoids showed differences from those of spinach, particularly the presence of a major 34 K band. This polypeptide occurs on reelectrophoresis of Chi and Chi a-b bands (Fig 4). 

Polypeptide Synthesis and Analysis. Sihca or controUed-pore glass supports treated with (chloromethyl)phenylethyltrimethoxysilane [68128-25-6] or its derivatives are replacing chloromethylated styrene—divinylbenzene (Merrifield resin) as supports in polypeptide synthesis. The sdylated support reacts with the triethyl ammonium salt of a protected amino acid. Once the initial amino acid residue has been coupled to the support, a variety of peptide synthesis methods can be used (34). At the completion of synthesis, the anchored peptide is separated from the support with hydrogen bromide in acetic acid (see Protein engineering Proteins). 

Figure 12.23 Hydropathy plots for the polypeptide chains L and M of the reaction center of Rhodobacter sphaeroides. A window of 19 amino acids was used with the hydrophohicity scales of Kyte and Doolittle. The hydropathy index is plotted against the tenth amino acid of the window. The positions of the transmembrane helices as found by subsequent x-ray analysis by the group of G. Feher, La Jolla, California, ate indicated by the green regions.

These predictive methods are very useful in many contexts for example, in the design of novel polypeptides for the identification of possible antigenic epitopes, in the analysis of common motifs in sequences that direct proteins into specific organelles (for instance, mitochondria), and to provide starting models for tertiary structure predictions. 

These results indicate that is it possible to change the fold of a protein by changing a restricted set of residues. They also confirm the validity of the rules for stability of helical folds that have been obtained by analysis of experimentally determined protein structures. One obvious impliction of this work is that it might be possible, by just changing a few residues in Janus, to design a mutant that flip-flops between a helical and p sheet structures. Such a polypeptide would be a very interesting model system for prions and other amyloid proteins. 

Amino acid analysis itself does not directly give the number of residues of each amino acid in a polypeptide, but it does give amounts from which the percentages or ratios of the various amino acids can be obtained (Table 5.2). If the molecular weight and the exact amount of the protein analyzed are known (or the number of amino acid residues per molecule is known), the molar ratios of amino acids in the protein can be calculated. Amino acid analysis provides no information on the order or sequence of amino acid residues in the polypeptide chain. Because the polypeptide chain is unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-termuial end. 

Later we return to an analysis of the 1° structure of proteins and the methodology used in determining the amino acid sequence of polypeptide chains, but let s first consider the extraordinary variety and functional diversity of these most interesting macromolecules. 

The standard protocol for analysis of the amino acid composition of proteins is discussed in Section 5.1. Results of such analyses allow the researcher to anticipate which methods of polypeptide fragmentation might be useful for the protein. 

End-group analysis reveals several things. First, it identifies the N- and C-ter-minal residues in the polypeptide chain. Second, it can be a clue to the number of ends in the protein. That is, if the protein consists of two or more different polypeptide chains, then more than one end group may be discovered, alerting the investigator to the presence of multiple polypeptides. 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Specificity of Representative Polypeptide Cleavage Procedures Used in Sequence Analysis... 

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