PER protein

FIGURE 5.23 Electrospmy mass spectrum of the protein, aerolysin K. The attachment of many protons per protein molecule (from less than 30 to more than 50 here) leads to a series of m/z peaks for this single protein. The inset shows a computer analysis of the data from this series of peaks that generates a single peak at the correct molecular mass of the protein. (Adapted from Figure 2 in Mann, M., and Wilm, M., 1995. Trends in Biochemical Sciences 20 219-224.)... 

First isolated from D. desulfuricans (28), desulfoferrodoxin (Dfe) was also isolated from D. vulgaris (29). D is a 28-kDa homodimer that contains two monomeric iron centers per protein. These iron centers were extensively characterized by UV/visible, EPR, resonance Raman, and Mossbauer spectroscopies (30). The data obtained were consistent with the presence of one Dx-like center (center I) and another monomeric iron center with higher coordination number (penta or hexacoordinate), with 0/N ligands and one or two cysteine residues (center II). Comparison of known Dfx sequences led to the conclusion that only five cysteines were conserved, and that only one of them could be a ligand of center II (31). 

Enzyme modified w. SC-PEG (5000) No. ofmPEGs linked per protein molecule % of native activity (Substrate )... 

The number of hydrazine groups per protein molecule can be determined by reacting a small portion of the hydrazine-modified protein with p-nitrobenzaldehyde, which forms a chromogenic product upon formation of the hydrazone derivative (Figure 1.111). 

Most references to the use of EDC describe the optimal reaction medium to be at a pH from 4.7 to 6.0. However, the carbodiimide reaction occurs effectively up to at least pH 7.5 without significant loss of yield. Conjugations done under mildly alkaline pH conditions (e.g., pH 8.5) also can be done to limit the polymerization of proteins, while still facilitating the coupling of a carboxylate-containing molecule at a low substitution level per protein. See Chapter 19,... 

A spectrophotometric assessment of the F/P ratio should be done after purification of the tagged antibody. The measurement of absorbance at 495 nm (for fluorescein) divided by the absorbance at 280 nm should be between 0.3 and 1.0 to obtain a good fluorescent derivative of acceptable activity and low background. This usually translates into a ratio of about 4-7 fluorescein molecules per protein molecule. 

Reactions done with NHS-PEG -biotin compounds typically are done with the reagent in molar excess over the amount of protein being modified. The efficiency of the reaction is dependent on the concentrations of reactants and the solvent exposed area of the amine groups on the protein. Reactions done with a 10-fold molar excess of NHS-PEG -biotin usually will result in at least 2-3 biotin labels per protein, while doubling the molar excess should provide 4-6 biotinylations. The optimal number of biotin groups added to a particular protein should be determined experimentally to provide the best performance in the intended application. 

In experiments23, we have observed a metrology-limited height of 20 pm per pixel, and an assay-limited height of 75 pm per protein spot. Based on these values, the scale-free minimum detectable height... 

Recent nutritional evaluations of cucurbit seeds have accentuated the currently popular Buffalo gourd ( . foetidissima) and are shown in Table III. PER (protein efficiency ratio) values of full-fat and natural and autoclaved fat-free meals fed to weanling... 

Chymotrypsin, in addition to its proteolytic activity, can also function as an esterase.1 It is inactivated by D.F.P., etc. (p. 186). The esterases firmly bind the phosphorus of D.F.P., and in the case of chymotrypsin the reaction is bimolecular, yielding a crystalline derivative containing two isopropoxy groups and one atom of phosphorus per protein molecule, but no fluorine.2... 

Multiple alignments of repeats are constructed in an iterative manner. The initial alignment is based on definitions from determined protein structures or else from the literature. In the initial database search step, a profile constructed from the multiple alignment is compared with a sequence database. Top scoring sequences are considered using complementary approaches such as PSI-BLAST and FASTA to provide the two thresholds minimum E value and minimum number of repeats per protein required. After one or two iterations, the final alignment and the thresholds are stored in the SMART database to allow the detection of repeats in any sequence. 

The mechanism of zinc deprivation by 3-nitrosobenzamide was elucidated most recently. When the reconstituted nucleocapsid protein p7 of HIV-1 (15 i-M) was incubated with 3-nitrosobenzamide (300 iM) at pH 7.5, three disulfide bonds per protein molecule were formed while 3-nitrosobenzamide was reduced to the hydroxylamine. Molecular masses of p7 adducts augmented by one or two 3-nitrosobenzamide residues were observed by electrospray ionization MS, consistent with covalent bond formation between cysteine sulfur and the nitroso nitrogen atom127. 

The calcium mediated contraction of smooth muscle, which unlike striated muscle does not contain troponin, is quite different and requires a particular calcium-binding protein called calmodulin. Calmodulin (CM) is a widely distributed regulatory protein able to bind, with high affinity, four Ca2+ per protein molecule. The calcium—calmodulin (CaCM) complex associates with, and activates, regulatory proteins, usually enzymes, in many different cell types in smooth muscle the target regulatory proteins are caldesmon (CDM) and the enzyme myosin light chain kinase (MLCK). As described below, CaCM impacts on both actin and myosin filaments. 

Lactamases of classes A, C, and D are serine peptidases and as such have been discussed in Sect. 3.3. Class B /1-lactamases, in contrast, are met-allohydrolases. For example, a class B /1-lactamase isolated from Bacillus cereus was shown to contain two Zn-atoms per protein molecule, of which only one is essential for catalysis. Three histidine residues act as ligands for the first Zn2+ ion, and a fourth histidine contributes to the binding of the second Zn-atom [82] [83],... 

Figure 1. In most examples of biological rhythms, sustained oscillations correspond to the evolution toward a hmit cycle. The limit cycle shown here was obtained in a model for circadian oscillations of the PER protein and per mRNA in Drosophila [107].

Molecular models for circadian rhythms were initially proposed [107] for circadian oscillations of the PER protein and its mRNA in Drosophila, the first organism for which detailed information on the oscillatory mechanism became available [100]. The case of circadian rhythms in Drosophila illustrates how the need to incorporate experimental advances leads to a progressive increase in the complexity of theoretical models. A first model governed by a set of five kinetic equations is shown in Fig. 3A it is based on the negative control exerted by the PER protein on the expression of the per gene [107]. Numerical simulations show that for appropriate parameter values, the steady state becomes unstable and limit cycle oscillations appear (Fig. 1). 

Another simplification can be made if all binding sites are independent (noncooperative binding) and they can be attributed to classes of identical sites. In protein-drug affinity studies the fraction of drug molecules bound (D4) per protein molecule (P) is given by... 

The mammalian molecular oscillator, similar to that in Drosophila and Neurospora, is composed of two interconnected feedback loops, one within the negative limb and one within the positive limb. The members of the positive limb (CLOCK and BMALl) activate transcription of the genes encoding the negative limb components (cryptochromes and period proteins). PER and CRY proteins then form heterotypic protein complexes that are translocated into the nucleus, and once these complexes reach a critical threshold level, they suppress the activity of CLOCK and BMALL As a consequence, the concentration of CRY and PER proteins falls below the threshold required for autorepression, and a new cycle of CryjPer transcription can initiate (for review, see Reppert Weaver 2002). 

CRY and PER proteins not only repress the activity of their own genes, but they also stimulate the expression of BMALl and CLOCK. How can the CRY and PER repressors activate the transcription of these positive hmb components The most likely scenario would imply a hitherto unknown repressor whose gene is under the negative control of CRYs and PERs. 

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