Resonance Raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

Proc. Natl. Acad. Sci. U.S.A.

Finkelstein

Kim

et al. 2007

104

Neuroglobin (Ngb), a protein in the globin family, is found in vertebrate brains. It binds oxygen reversibly. Compared with myoglobin (Mb), the amino acid sequence has limited similarity, but key residues around the heme and the classical globin fold are conserved in Ngb. The CO adduct of Ngb displays two CO absorption bands in the IR spectrum, referred to as N 3 (distal histidine in the pocket) and N 0 (distal histidine swung out of the pocket), which have absorption spectra that are almost identical with the Mb mutants L29F and H64V, respectively. The Mb mutants mimic the heme pocket structures of the corresponding Ngb conformers. The equilibrium protein dynamics for the CO adduct of Ngb are investigated by using ultrafast 2D-IR vibrational echo spectroscopy by observing the CO vibration's spectral diffusion (2D-IR spectra time dependence) and comparing the results with those for the Mb mutants. Although the heme pocket structure and the CO FTIR peak positions of Ngb are similar to those of the mutant Mb proteins, the 2D-IR results demonstrate that the fast structural fluctuations of Ngb are significantly slower than those of the mutant Mbs. The results may also provide some insights into the nature of the energy landscape in the vicinity of the folded protein free energy minimum.myoglobin mutants ͉ protein dynamics ͉ energy landscape N euroglobin (Ngb) is a recently discovered family of vertebrate globin proteins. Ngb is expressed predominantly in nerve tissue. It has been hypothesized that Ngb facilitates O 2 diffusion to protect neuronal cells from hypoxia and ischemia (1). Ngb concentration in the brain is fairly low, too low to play the role that myoglobin (Mb) plays in the red muscles. However, because the expression level of Ngb is enhanced under hypoxic conditions in vitro as well as ischemia in vivo (2, 3), Ngb may be involved in neuronal responses to ischemia. A much higher concentration of Ngb is found in the retina. The concentration is high enough to deliver O 2 to mitochondria (4) and may facilitate O 2 diffusion in a manner similar to Mb. Another possibility is that Ngb is involved in redox-coupled sensor regulation for signal transduction in the brain. Ngb binds to the GDP-bound form of the ␣-subunit of heterotrimeric G protein under oxidative stress (5).The 3D structures of Ngb from human and mouse, both having 151 amino acids, have been published (6, 7). Comparisons of Ngb with vertebrate Mb and Hb sequences show only minor similarities at the amino acid level (1), but Ngb features the conserved classical globin fold and contains heme (6). The heme iron in both the ferrous and ferric forms of Ngb is hexacoordinated (8), in contrast to mammalian Mb and Hb, which contain pentacoordinated heme iron. Although hexacoordinated heme has been reported in plant, bacteria, and invertebrate globins, its physiological significance is not yet understood (9). In Ngb, an external gaseous ligand must compete with the sixth ligand, the distal histidine (E7 in helix notation), for binding. Several key residues...

Section: Resultsmentioning

confidence: 99%

“…1). Experiments, theory, and molecular dynamics simulations have shown that both the local and global coupling between protein structure and the CO vibrational transition frequency is mainly through the electric field along the CO bond (19,20,24,29,30,(35)(36)(37). The protein residues range from charged, to polar, to nonpolar.…”

Section: Resultsmentioning

confidence: 99%

Neuroglobin dynamics observed with ultrafast 2D-IR vibrational echo spectroscopy

Proc. Natl. Acad. Sci. U.S.A.

Finkelstein

Kim

et al. 2007

104

“…In the spectrum of wt-Ngb (solid curve), there are two CO bands at 1,932 cm Ϫ1 and 1,968 cm Ϫ1 . These have been called the N 3 (lower frequency) and N 0 (higher frequency) conformers, respectively, in analogy to the A 3 and A 0 bands of Mb (26)(27)(28). The significant width of the CO-stretching bands of Ngb and their Gaussian shapes implies that there is structural heterogeneity associated with each protein substate.…”

Section: Resultsmentioning

confidence: 99%

Disulfide bond influence on protein structural dynamics probed with 2D-IR vibrational echo spectroscopy

Proc. Natl. Acad. Sci. U.S.A.

Kim

Kwak

et al. 2007

Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to Ϸ100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the protein's structure.FTIR ͉ neuroglobin ͉ ultrafast 2D-IR

“…Carbonmonoxy-myoglobin (MbCO) has been extensively studied both experimentally [47][48][49][50][51][52][53][54][55][56] and computationally. [57][58][59][60] Myoglobin (Mb) is a small globular heme protein weighing approximately 17 kDa that is found in mammalian muscle tissue.…”

Section: D Vibrational Echo Experiments a Illustration Of The mentioning

confidence: 99%

Probing dynamics of complex molecular systems with ultrafast 2D IR vibrational echo spectroscopy

Finkelstein

Zheng

Phys. Chem. Chem. Phys.

et al. 2007

148

Ultrafast 2D IR vibrational echo spectroscopy is described and a number of experimental examples are given. Details of the experimental method including the pulse sequence, heterodyne detection, and determination of the absorptive component of the 2D spectrum are outlined. As an initial example, the 2D spectrum of the stretching mode of CO bound to the protein myoglobin (MbCO) is presented. The time dependence of the 2D spectrum of MbCO, which is caused by protein structural evolution, is presented and its relationship to the frequency-frequency correlation function is described and used to make protein structural assignments based on comparisons to molecular dynamics simulations. The 2D vibrational echo experiments on the protein horseradish peroxidase are presented. The time dependence of the 2D spectra of the enzyme in the free form and with a substrate bound at the active site are compared and used to examine the influence of substrate binding on the protein's structural dynamics. The application of 2D vibrational echo spectroscopy to the study of chemical exchange under thermal equilibrium conditions is described. 2D vibrational echo chemical exchange spectroscopy is applied to the study of formation and dissociation of organic solute-solvent complexes and to the isomerization around a carbon-carbon single bond of an ethane derivative.