Apo-proteins

Prosthetic groups and cofactors, whether organic or metallic, may be removed from a protein to create an inactive apo protein or enzyme. Loss of these groups may occur through environmental changes, such as removing metal ions from solution or adding denaturants to unfold... 

In mammals a single PPTase is used for the posttranslational modification of three different apo-proteins the carrier proteins of mitochondrial and cytosolic FASs and the aminoadipate semialdehyde reductase implicated in lysine degradation. The crystal structure of human PPT ase has been determined and found to be most closely related to the class II Sfp-like enzymes. Architectural and mechanistic differences between the type II human PPTase and the type I bacterial PPTases include a divalent cation coordinated by the a-phosphate of CoA, a Glu and an Asp residue, and three water ligands in type I PPTases versus a divalent cation coordinated by a- and /3-phosphates of CoA, two to three protein side chains, and a water molecule in the human PPT ase. 

A more complete picture of the protein-Ugand interaction is given by the docking mode of the bioactive conformation in the binding pocket of the receptor protein. In case the apo protein structure is known from NMR or... 

The PLIMSTEX curve for 0.3 pM WT-IFABP titrated with potassium oleate fits well with a 1 1 binding model [22, 24], The K and ADi (difference between the average deuterium level of one-ligand-bound protein and that of apo protein) for WT-IFABP are (2.6 + 0.6) x lO and 13.8 + 0.7 (Table 11.1), respectively, indicating that a strong interaction between oleate and WT-IFABP occurs with protection of 14 backbone amide hydrogens. 

One asset of mass spectrometry in protein science is that ESI and MALDI [11, 75] can introduce noncovalent complexes to the gas phase [12, 76, 77]. If one can assume that the gas-phase ion abundances (peak intensities) for the complex, apo protein, and ligand are directly related to their equilibrium concentrations in solution, the relative and absolute binding affinities can be deduced [78-81]. Extended methods are now available that also make use of the intensity of the complex and the protein at high ligand concentration to determine binding constants [78, 82-84]. 

Perhaps the most fundamental fimctional property of a heme prosthetic group at the active site of a heme protein is the relative stability of the reduced and oxidized states of the heme iron. A number of structural characteristics of the heme binding environment provided by the apo-protein have been identified as contributing to the regulation of this equilibrium and have been reviewed elsewhere 82-84). Although a comprehensive discussion of these factors is not possible in the space available here, they can be summarized briefly. The two most significant influences of the reduction potential of the heme iron appear to be the dielectric constant of the heme environment 81, 83) and the chemical... 

Fig. 6. Deconvolved amide F region FTIR spectra of apo- and heme-hemopexin. The amide F FTIR spectra of apo- and heme-hemopexin in D2 O were recorded and curve-fitted to resolve the individual bands. The differences between the original and fitted curves are shown in the upper traces in the panels. The estimated helix (15%), beta (54%), turn (19%), and coil (12%) content of the apo-protein are not significantly changed upon heme binding 104). This analysis was required because of the positive 231-nm elhpticity band in hemopexin and is consistent with the derived crystal structure results.

Fig. 14. Effects of temperature on the absorbance of hemopexin and the N-domain of hemopexin. The unfolding of hemopexin and N-domain in 25 mM sodium phosphate, pH 7.4, was examined using absorbance spectroscopy (N. Shipulina et al., unpublished). The second derivative UV absorbance spectra of the protein moieties were used to follow protein unfolding and the Soret and visible region spectra to monitor the integrity of the heme complexes, as done with cytochrome 6502 (166). The ferri-heme complex is more stable than the apo-protein moiety, but the is slightly lower than that assessed by DSC, indicating that changes in conformation occur before thermodynamic unfolding. Reduction causes a large decrease in heme-complex stabihty, which is proposed to be a major factor in heme release from hemopexin by its cell membrane receptor, and addition of 150 mM sodium chloride enhanced the stabihty of ah forms of hemopexin.

Methods to electrically wire redox proteins with electrodes by the reconstitution of apo-proteins on relay-cofactor units were discussed. Similarly, the application of conductive nanoelements, such as metallic nanoparticles or carbon nanotubes, provided an effective means to communicate the redox centers of proteins with electrodes, and to electrically activate their biocatalytic functions. These fundamental paradigms for the electrical contact of redox enzymes with electrodes were used to develop amperometric sensors and biofuel cells as bioelectronic devices. 

Crystallization experiments using rALBP were immediately successful. With seeding, octahedral crystals of the apo-protein grew to a length of 0.4 mm and a height of 0.3 mm. These crystals give diffraction data to 2.4 A. An entire data set was collected to 2.7-A resolution using the area detector system. Statistical details of the combined X-ray data set are presented in Table 8.1. 

Zayats M, Katz E, Willner I. Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor. Journal of the American Chemical Society 2002, 124, 2120-2121. 

F3. Fairnaru, M., Glangeaud, M. C., and Eisenberg, S., Radioimmunoassay of human high density lipoprotein apo-protein A-I. Biochim. Biophys. Acta 386, 432-443 (1975). 

Visual excitation (1) in both vertebrates and invertebrates is initiated via light absorption by visual pigments consisting of a chromophore covalently bound to an apo-protein, opsin. Biochemical extraction studies have shown that in all pigments the chromophore is a Schiff base derivative of 11-cis retinal (Fig. 

The cell surface receptors also remove the carboxy terminal arginine residue from RBP, thus inactivating it by reducing its affinity for both transthyretin and retinol. As a result, apo-RBP is filtered at the glomerulus. Some may be lost in the urine, but most is resorbed in the proximal renal tubules and is then catabolized by lysosomal hydrolases. This seems to be the main route for catabolism of RBP the apo-protein is not recycled (Peterson et al., 1974). 

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