Upon photodissociation of its ligand, COHbA exhibits a wide range of nonequilibrium relaxation phenomena that start within a fraction of a picosecond and extend out to tens of microseconds. In addition, equilibrium fluctuations of the protein result in conformational averaging. All of these dynamics can have an impact on ligand rebinding. In an effort to better understand the relationship between conformational dynamics and ligand-binding reactivity, COHbA was embedded in a room temperature trehalose sugar glass (Hagen et al. Science 1995, 269, 959) in order to uncouple solvent motions from protein dynamics as well as reduce the amplitude of large-scale protein conformational fluctuations. Time-resolved resonance Raman spectroscopy and ligand-rebinding kinetics show that the trehalose glass does not impede the initial fast relaxation of the iron-histidine linkage, but does dramatically impede conformational averaging and completely eliminates ligand escape at all temperatures from 140 K to room temperature. Fluorescence measurements indicate that in the trehalose glass the picosecond tryptophan lifetimes are nearly unchanged, but there is a complete absence of the nanosecond fluorescence decay (observed in aqueous solutions), which is replaced by a decay of ∼700 ps. This change in the fluorescence decay is ascribed to a significant decrease in the structural dynamics that normally allow transient opening of the distal heme pocket.

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Among various sounds in the environment, natural sounds, such as water sounds and birdsongs, have proven to be highly preferred by humans, but the reasons for these preferences have not been thoroughly researched. This paper explores differences between various natural and urban environmental sounds from the viewpoint of objective measures, especially psychoacoustical parameters. The sound samples used in this study include the recordings of single sound source categories of water, wind, birdsongs, and urban sounds including street music, mechanical sounds, and traffic noise. The samples are analyzed with a number of existing psychoacoustical parameter algorithmic models. Based on hierarchical cluster and principal components analyses of the calculated results, a series of differences has been shown among different sound types in terms of key psychoacoustical parameters. While different sound categories cannot be identified using any single acoustical and psychoacoustical parameter, identification can be made with a group of parameters, as analyzed with artificial neural networks and discriminant functions in this paper. For artificial neural networks, correlations between network predictions and targets using the average and standard deviation data of psychoacoustical parameters as inputs are above 0.95 for the three natural sound categories and above 0.90 for the urban sound category. For sound identification/classification, key parameters are fluctuation strength, loudness, and sharpness.

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.

The pH-induced unfolding and refolding of the carbon monoxide-bound derivative of horse skeletal myoglobin (COMb) encapsulated in porous sol−gels is probed using several optical techniques in conjunction with different unfolding/refolding protocols. UV resonance Raman (UVRR) spectroscopy and fluorescence are used to monitor the unfolding of the globin and exposure of the A helix to solvent. Absorption spectra, visible resonance Raman (VRR) spectra, and geminate recombination are used to probe the heme and the heme environment. Encapsulation slows the kinetics of acid-induced unfolding and dramatically slows the kinetics of refolding. The spectra and the kinetics imply that this approach allows for the detailed study of the burst phase of unfolding. Using different encapsulation protocols and sequences of solvent replacements, it is possible to trap and probe not only low-pH forms observed in solution-phase studies but also novel partially unfolded species that are likely to be important unfolding and folding intermediates. The role of water as a chaotropic agent is indicated by the spectral changes that occur in the introduction and subsequent removal of glycerol from the solution bathing the unfolded and partially unfolded, sol−gel encapsulated COMb. The results directly support the view that unfolding or increasing the exposure to solvent of at least some segment of the A helix is the initial step in the unfolding pathway. In addition, the results indicate that the refolding of the A helix is likely to be the last process in the refolding pathway.