Rearrangements in the Protein Structure

FIGURE 15.19 Interdependency of the mqor subprocesses that are involved in the overall protein adsorption process. Adsorption promotion is denoted by a + sign and adsorption 

6 REVERSIBILITY OF THE PROTEIN ADSORPTION PROCESS DESORPTION AND EXCHANGE 

Phenomenologically, a system is in equilibrium if no changes take place at constant surroundings. At constant pressure p and temperature T, the equilibrium state of a system is characterized by a minimum value of the total Gibbs energy G. Any other 

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The 3D structure of a native protein (in aqueous solution) is only marginally thermodynamically stable and it is sensitive to changes in its environment. It is, therefore, not surprising that adsorption is often accompanied by rearrangements in the protein s 3D structure. It is commonly observed experimentally that the thickness of an adsorbed protein layer is comparable to the dimensions of the protein molecule in solution. It indicates that the adsorbed protein molecules remain compactly structured. 

Evidence for domain recruitment has been identified in a wide variety of proteins [47], mechanistically ranging from simple N- or C-terminal fusion to multiple internal insertions and possibly circular permutations [48]. A recent analysis of proteins in the protein structure database (PDB) has further indicated that structural rearrangements as a result of domain shuffling have significantly contributed to today s functional diversity [49]. A brief overview of the various modes of domain recruitment and their effects on function, is presented on examples of /lex-barrel structures. 

In x-ray crystallographic analysis of COMb it has been noted that there is no pathway for the migration of CO between the buried binding site and solvent. Therefore, rapid rearrangements of the protein structures should be accompanied by recombination of CO. The transient absorption study on the relaxation process of photolysis product of COMb revealed that the recombination of the photodissociated CO was nonexponential. Fe-CO stretching band that appears in Raman spectra, is a good maker for this distal pocket environment. We pursued the time profile of this band to monitor the dynamics and found an intermediate species common to all the mutants. We will discuss the existence of the intermediate which correspond to "open form". 

For HPA, removed from the sorbents hematite, silica and polyoxymethylene, the molecular structure has been compared with that of the native molecule, on the basis of their circular dichroism spectra (11). It was found that after desorption the helix content of HPA is some twenty to thirty percent lower. This reduction is virtually independent of the type of sorbent and the desorption method. It suggests that the change in the helix content is related to properties of the protein molecule itself. It is still not clear to which extent the adsorption and the desorption step affect the protein structure. It Is furthermore interesting that the helix reduction is larger for the samples with lower F -values. This supports the earlier conclusion that a reduced Fp value reflects further structural rearrangements in the protein molecule. It is noted that the decrease In the helix content of desorbed HPA found by us is considerably less than that reported by others (19). 

Overall the structures are well preserved (rmsd 2-3 A), even on ns time scales, without any ad hoc restraints imposed in the systems. Local structural changes are observed, both in terms of side chain rearrangements in the protein-DNA interface, and in flexible loop regions. The DNA structure is also well behaved, and seems to be stabilized by the presence of the protein, but bending and twisting of the DNA has been observed in several cases, especially in the complexes where the DNA has a canonical B-type conformation. 

The Ca -ATPase has been crystallized in both conformations [119,152-155]. The two crystal forms are quite different [10,88-93,156-161], suggesting significant differences between the interactions of Ca -ATPase in the Ei and E2 conformations. Since the Ei-E2-transition does not involve changes in the circular dichroism spectrum of the Ca -ATPase [162], the structural differences between the two states presumably arise by hinge-like or sliding motions of domains rather than by a rearrangement of the secondary structure of the protein. 

Clearly, the spectroscopic properties of the P clusters in the proteins do not reveal their structural nature. However, extrusion of these clusters from the protein leads to the clear identification of 3-4 Fe S clusters(13.291. Despite the uncertainties inherent in the extrusion procedure (due to possible cluster rearrangement) the extrusion result supports the Dominant Hypothesis, which designates the P centers as Fe S units, albeit highly unusual ones. The P clusters are thought to be involved in electron transfer and storage presumably providing a reservoir of low potential electrons to be used by the M center (FeMo-co) in substrate reduction. 

All proteins and their cofactors show complex spectra in the midIR region 1000-1800 cm-1. Typical midIR absorbers are 0=0, C=C, C-C, N-H stretches, and their change in precise spectral position allows us to follow the structural rearrangement of the protein and cofactor in time. Good examples are the transfer of charge, the rearrangement of H-bonds and cis-trans isomerization of a chromophore (like in PYP). Moreover, polarized measurements allow determination of the dynamic reorientation of these chromophores. 

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