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The formation of amyloid aggregates in human organs and tissues causes the development of incurable diseases. However, experimental studies of the mechanism of amyloid formation by proteins and the structural characteristics of amyloids are complicated because of the heterogeneity and high molecular weight of the aggregates. We used limited proteolysis and mass spectrometry for the identification of regions in the apomyoglobin polypeptide chain, which give rise to intermolecular interactions in amyloid structures. Tandem mass spectroscopy enabled the identification of regions in the myoglobin polypeptide chain, which form the core of amyloid structures. It was shown that the main structural elements for the formation of the core of amyloid fibrils in myoglobin were regions from 60 through 90 and from 97 through 124 amino acid residues. These regions coincide well with those theoretically predicted. This approach yielded important data on the structure of protein molecules in aggregates and on conformational rearrangements of apomyoglobin upon amyloid formation. Keywords: amyloids, apomyoglobin, limited proteolysis, amyloidogenic region, HPLC-MS/MS, identification of peptides, INTRODUCTION To perform its function, protein must acquire a unique spatial structure in the process of folding. However, such condition may occur in a cell, under which protein molecules are [...]

The effects of small viscogenic cosolvents and folding and unfolding kinetics of the apomyoglobin pH 4 molten globule intermediate were investigated. It was found that, in both folding and unfolding conditions, the apparent rate constant decreases when solvent viscosity increases.

A molecular dynamics study is conducted to analyze the correlations between the nuclear volume, solvent-accessible surface and the folding state of various unfolded conformers of a [alpha] or a [beta] protein with any helical peptide. The results prove that the unfolded conformations need not be necessarily extended, as the volume variation does not affect the folding state to a large extent and hence is ignored in the study of the interiors of the protein.

The focus of the study is on requirements for a myoglobin (Mb) hemoglobin (Hb) functional model in aqueous solution, discrimination between [O.sub.2] and CO by hemoCDI, and the mechanism for autoxidation of oxy-hemoCDI. The results revealed the reason why hemoCDI acts as an excellent functional model for Mb and Hb that works in aqueous solution.

Covalently linked films of the ferric heme protein myoglobin and poly-L-lysine graphite electrodes reacted with tert-butylhydroperoxide (tBuOOH) to form ferryloxy protein species according to Michaelis-Menten enzyme kinetics. Apparent kinetic constants are most likely governed by acidity-controlled protein conformations and their binding with tBuOOH in the intermediate protein-substrate complex.

The preparation of reconstituted myoglobin with the cobalt porphycene and O(sub 2) kinetics are presented. It is revealed that the constituted myoglobin with the cobalt porphycene has a high O(sub 2)affinity caused by its slow O(sub 2) dissociation.

It is for the first time that orientational probability distribution function for CO transiently docked in myoglobin (Mb) and Hb is established. The ordered protein structure around the active site of the enzyme has a selectivity even for small ligands.

Spectrally resolved infrared stimulated vibrational echo data were obtained for sperm whale carbonmonoxymyoglobin (MbCO) at 300 K. The dephasing dynamics of the CO ligand were measured and compared with the dephasing dynamics calculated with molecular dynamics simulations for MbCO with the residue histidine-64 (His64). Results show that the dynamics of the His64 can be considered as a major source of dephasing for the CO ligand.

Solution (Super 1)H NMR was used to elucidate the magnetic properties and electronic structure of the prosthetic group in high-spin, ferrous deoxy myoglobin from the sea hare Aplysia limacina. Understanding of the NMR spectra of high-spin ferrous hemoproteins requires the determination of the anisotropy and orientation of the paramagnetic susceptibility tensor from a variety of proteins with varied active site geometry.

The genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in Escherichia coli is described by evolving an orthogonal tTNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical ' handle' for fluorescent labeling of p-Tpa-encoded myoglobin by the photoclick reaction. The structural basis for the biosynthetic incorporation of p-Tpa into proteins is analyzed by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa.

A conserved 2-His-1-Glu metal center, which is found in natural nonheme iron-containing enzymes, is engineered into sperm whale myoglobin by replacing by replacing Leu29 and Phe43 with Glu and His. The analysis has presented a novel protein model of NOR and has provided insights into a newly discovered member of the NOR family, gNOR.

GuHCl-induced, equilibrium unfolding of five sperm whale metMb variants are measured to understand the discrepancies during the unfolding of wild-type holomyoglobin in the ferric state (metMb). The high affinities of native apoMb (N) for hemin are found to have significant influence on the stability of holo-metMb and the partially unfolded intermediate with hemin bound ((IH) exhibiting a hemichrome spectra are indicative of a bis-histidyl axial coordination which is seen clearly when the stability of the N state or its affinity for hemin is reduced.

Correlated single-crystal spectroscopy with crystallography was employed to characterize the single crystal of the nitrite adduct of ferric myoglobin (Mb) exposed to high-intensity synchrotron X-ray radiation. The 1.55 [Angstrom] resolution crystal structure of the photoproduct reveals retention of the O-binding mode for binding of nitrite to the iron center.

A pair of myoglobins containing inherently distorted [alpha]-ethyl-2,4-dimethyldeuteroheme or undistorted 2,4-dimethyldeuteroheme were prepared to study the functional consequence of intrinsic heme deformation. A distinctive controlling mechanism observed for ligand binding by the deformed heme revealed that the iron atom is more attracted by the proximal histidine to reduce the affinity of exogenous ligands for the ferrous heme.

Backbone-modified phosphorothioate DNA and biotin- and maleimide-containing bifunctional linkers are used to achieve controlled alignment of streptavidin (STV), myoglobin, and nanoparticles with nanometer resolution. The proposed method which facilitates the high yield, nanoscale-precision conjugation of multiple proteins to DNA could be used to design a linker to conjugate any proteins and nanomaterials specifically for a wide range of applications.

The pathways identified by alkyl isocyanides (CNRs) for diatomic ligand movement into and out of Mb, with their side chains acting as transition state analogues are reviewed. The strong correlation observed between the fraction of in conformer, [F.sub.in], for long-chain MbCNR complexes, the fraction of geminate recombination of photodissociated [O.sub.2], and the bimolecular rates of [O.sub.2] entry into the distal pocket revealed that alkyl isocyanides serve as transition state analogues for the movement of [O.sub.2] into and out of the binding pocket of Mb.

A study is conducted to gain insight into the low- and high-frequency peaks observed for the alkyl isocyanides upon binding to sperm whale myoglobin (Mb). The internal rebinding for in alkyl side chain conformation is promoted by the photodissociated ligand which is stuck in the distal pocket while the out conformation that inhibited geminate recombination is attributed to the part of the ligand that is already in an open E7 channel, poised for rapid escape.

A strategy is described by which reactive binding of a weakly bound, 'dynamically docked (DD)', complex without a known structure is strengthened electrostatically through optimized placement of surface charges, and its utility in modulating complex formation between myoglobin (Mb) and cytochrome [b.sub.5] ([b.sub.5]) is described. The measurements have shown that the presence of fast photoinduced singlet-electron transfer (ET) across the protein-protein interface.

Strongly electron-withdrawing perfluoromethyl (C[F.sub.3]) groups are substituted as heme side chains of myoglobin (Mb) to obtain larger alterations of the heme electron structure in order to analyze the relationship between the [O.sub.2] affinity of Mb and the electronic properties of heme peripheral side chains. The introduction of electron-withdrawing groups has increased the preferential binding of CO to the protein over that of [O.sub.2].

The effects of solvation on proteins dynamics are examined by using molecular dynamics simulations and myoglobin as a model system. The studies have shown that water is able to interact with proteins in diverse ways, which has led to different kinds of perturbations in their intrinsic dynamic behavior.

The critical contribution of the distal histidine towards controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases is highlighted. The two conformations of His55 in equilibrium at room temperature is found to provide a level of control that allows the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.

A study was conducted to examine the role of copper in regulation of ligand binding in [Cu.sub.B]Mb, a mutant of sperm whale myoglobin as a model for cytochrome c oxidase (CcO). The data demonstrated that the [Cu.sub.B]Mb/[Cu.sup.+] complex reproduces essential structural and kinetic features of CcO very much similar to the complex in Thermus thermophilus CcO (TtCcO) than to bovine CcO (bCcO).

Several studies are conducted to explain the network of pathways involved in the diffusion of the CO molecule inside the myoglobin protein. The entire binding process is shown to be highly dependent and affected by the various cavities present inside the protein.

Myoglobin (Mb) and horseradish peroxidase (HRP) are both reconstituted with a meso-unsubstituted iron corrole and their electronic configurations and peroxidase activities are examined. The catalytic activities of both proteins toward guaiacol oxidation are different and this finding is attributed to the difference in the oxidation states of the corrole iron when these proteins are in the resting state.

Horse myoglobin is used as a model system for assessing suitability of amide hydrogen/deuterium exchange (HDX)- mass spectrometry (MS) with top-down electron capture dissociation (ECD) for analyzing structural transitions of the 17 kDa protein. The results have shown that the level of structural information obtainable with top-down ECD for small to medium-sized proteins have surpassed the traditional HDX-MS experiments, while at the same time has reduced the undesirable amide back exchange.

The studies have shown that water is detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not observed in the corresponding crystal structures. The spectral perturbation of pentacoordinate heme has offered a robust system for measuring nonspecific hydration of the active sites of heme proteins.

The studies have shown that the 221 [cm.sup.-1] mode of the photoproduct of the inactive P420 form of inducible nitric oxide synthase ([iNOS.subP420]) has not exhibited any [H.sub.2]O-[D.sub.2]O solvent isotope shift such as that found in the iron-histidine stretching mode of myoglobin, indicating that the proximal ligand of [iNOS.sup.P420] is not a histidine. The studies have supported the scenario that [iNOS.sub.P420] is inactivated by protonation of the native proximal thiolate ligand to a neutral thiol, instead of by ligand switching to a histidine.

Several molecular dynamics simulation studies are conducted to explain the breathing motions of myoglobin, a respiratory protein. The various key residues that work as switches between cavities are also identified.

A systematic global mapping of water motions in the hydration layer around a model protein of apomyoglobin in both native and molten globule states is reported. The results provide insight into a series of correlations of hydrating water dynamics and coupled fluctuations with local protein's chemical and structural properties which are significant for various water behaviors in the hydration layer with wide heterogeneity.

CO escaping pathways of Myoglobin are examined in terms of three-dimensional (3D) distribution function that is calculated by the 3D-RISM theory. The partial molar volume changes through the CO escaping pathways have agreed with those from the experiment.

Resonance Raman spectroscopy is used for characterizing heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives, the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. The lack of an effect on the key vibrational parameters of the Fe-[N.sub.His], Fe-CO and Fe-CN fragments is consistent with the earlier equilibrium and kinetic studies that have documented virtually identical functional properties for the native and reversed forms.

The nature of the Fe-[O.sub.2] bonding in oxy-myoglobin is examined by hybrid quantum mechanical/molecular mechanical calculations and gas-phase calculations using density functional theory (DFT). The protein has strengthened the [Fe.sup.III]-[O.sub.2.sup.-] character of the complex, thus affecting its bonding features and the oxygen binding affinity of Mb.

A photoacoustic calorimetry and transient absorption studies are conducted to examine the impact of substrate binding on the conformational dynamics associated with photodissociation of CO from [Fe.sup.2+] dehaloperoxidase (DHP) from Amphitrite ornata. The conformational changes observed for dissociation of ligand from DHP which significantly differed from that of photodissociation of CO from the structural homologue myoglobin have revealed that the structural dynamics in DHP are fine-tuned to enhance the peroxidase function of this protein.