Isolation of a myoglobin molten globule by selective cobalt(III)-induced unfolding

Reaction of the Schiff-base complex [Co(acetylacetonate-ethylenediimine)(NH3)2]+ with metmyoglobin at pH 6.5 yields a partially folded protein containing six Co(III) complexes. Although half of its α-helical secondary structure is retained, absorption and CD spectra indicate that the tertiary structure in both B-F and AGH domains is disrupted in the partially folded protein. In analogy to proton-induced unfolding, it is likely that the loss of tertiary structure is triggered by metal-ion binding to histidines. Cobalt(III)-induced unfolding of myoglobin is unique in its selectivity (other proteins are unaffected) and in allowing the isolation of the partially folded macromolecule (the protein does not refold or aggregate upon removal of free denaturant).

Structural factors controlling ligand binding to myoglobin: A kinetic hole-burning study

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CO-bound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as “kinetic hole burning.” The study of these correlations gives direct insights into the structure–function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates influence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.

In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1.34 × 104 M−1 s−1 (≈25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 106 M−1 s−1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 × 10−6 M−1 s−1 vs. 22.3 × 10−6 M−1 s−1; koff, 24.3 s−1 vs. 24.2 s−1; KO2, 0.91 × 10−6 M−1 vs. 0.92 × 10−6 M−1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 × 10−6 M−1s−1 vs. 0.51 × 10−6 M−1s−1; koff, 5.08 s−1 vs. 3.51 s−1; KCO, 1.77 × 10−7 M−1 vs. 1.45 × 10−7 M−1). All four substitutions are in the heme pocket and within 5 Å of the heme group.

Vibrational population relaxation of carbon monoxide in the heme pocket of photolyzed carbonmonoxy myoglobin: Comparison of time-resolved mid-IR absorbance experiments and molecular dynamics simulations

The vibrational energy relaxation of carbon monoxide in the heme pocket of sperm whale myoglobin was studied by using molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the δ- and ɛ-tautomers of the distal His-64. Vibrational population relaxation times of 335 ± 115 ps for the δ-tautomer and 640 ± 185 ps for the ɛ-tautomer were estimated by using the Landau–Teller model. Normal mode analysis was used to identify those protein residues that act as the primary “doorway” modes in the vibrational relaxation of the oscillator. Although the CO relaxation rates in both the ɛ- and δ-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the δ-tautomer with the distal His playing an important role in the energy relaxation mechanism. Time-resolved mid-IR absorbance measurements were performed on photolyzed carbonmonoxy hemoglobin (Hb13CO). From these measurements, a T1 time of 600 ± 150 ps was determined. The simulation and experimental estimates are compared and discussed.

The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin

The grail of protein science is the connection between structure and function. For myoglobin (Mb) this goal is close. Described as only a passive dioxygen storage protein in texts, we argue here that Mb is actually an allosteric enzyme that can catalyze reactions among small molecules. Studies of the structural, spectroscopic, and kinetic properties of Mb lead to a model that relates structure, energy landscape, dynamics, and function. Mb functions as a miniature chemical reactor, concentrating and orienting diatomic molecules such as NO, CO, O2, and H2O2 in highly conserved internal cavities. Reactions can be controlled because Mb exists in distinct taxonomic substates with different catalytic properties and connectivities of internal cavities.

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, Dl for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 × 10−7 cm2/s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, ≈1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Diffractive optics-based heterodyne-detected four-wave mixing signals of protein motion: From “protein quakes” to ligand escape for myoglobin

Ligand transport through myoglobin (Mb) has been observed by using optically heterodyne-detected transient grating spectroscopy. Experimental implementation using diffractive optics has provided unprecedented sensitivity for the study of protein motions by enabling the passive phase locking of the four beams that constitute the experiment, and an unambiguous separation of the Real and Imaginary parts of the signal. Ligand photodissociation of carboxymyoglobin (MbCO) induces a sequence of events involving the relaxation of the protein structure to accommodate ligand escape. These motions show up in the Real part of the signal. The ligand (CO) transport process involves an initial, small amplitude, change in volume, reflecting the transit time of the ligand through the protein, followed by a significantly larger volume change with ligand escape to the surrounding water. The latter process is well described by a single exponential process of 725 ± 15 ns at room temperature. The overall dynamics provide a distinctive signature that can be understood in the context of segmental protein fluctuations that aid ligand escape via a few specific cavities, and they suggest the existence of discrete escape pathways.

X-ray structure determination of a metastable state of carbonmonoxy myoglobin after photodissociation.

The x-ray structure of carbon monoxide (CO)-ligated myoglobin illuminated during data collection by a laser diode at the wavelength lambda = 690 nm has been determined to a resolution of 1.7 A at T = 36 K. For comparison, we also measured data sets of deoxymyoglobin and CO-ligated myoglobin. In the photon-induced structure the electron density associated with the CO ligand can be described by a tube extending from the iron into the heme pocket over more than 4 A. This density can be interpreted by two discrete positions of the CO molecule. One is close to the heme iron and can be identified to be bound CO. In the second, the CO is dissociated from the heme iron and lies on top of pyrrole ring C. At our experimental conditions the overall structure of myoglobin in the metastable state is close to the structure of a CO-ligated molecule. However, the iron has essentially relaxed into the position of deoxymyoglobin. We compare our results with those of Schlichting el al. [Schlichting, I., Berendzen, J., Phillips, G. N., Jr., & Sweet, R. M. (1994) Nature 317, 808-812], who worked with the myoglobin mutant (D122N) that crystallizes in the space group P6 and Teng et al. [Teng, T. Y., Srajer, V. & Moffat, K. (1994) Nat. Struct. Biol. 1, 701-705], who used native myoglobin crystals of the space group P2(1). Possible reasons for the structural differences are discussed.

Hydrated myoglobin's anharmonic fluctuations are not primarily due to dihedral transitions.

To characterize the functionally important anharmonic motions of proteins, simulations of carboxymyoglobin (MbCO) dynamics have been performed during which dihedral transitions were prohibited. Comparison of torsionally restrained and unrestrained protein dynamics simulated at three levels of hydration and at temperatures ranging from 100 to 400 K suggests that hydration "catalyzes" protein mobility by facilitating collective anharmonic motions that do not require dihedral transitions. When dihedral transitions were prohibited, dehydrated MbCO, to a good approximation, exhibited only harmonic fluctuations, whereas hydrated MbCO exhibited both harmonic and anharmonic motions. The fluctuation of helix centers of mass also remained highly anharmonic in the torsionally restrained hydrated system. Atomic mean-square fluctuation at 300 K was reduced upon prohibition of dihedral transitions by only 28% and 10% for MbCO hydrated by 350 and 3830 water molecules, respectively.

Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate

On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (i) truncate hydrophobic side chains in native helix packing sites, (ii) truncate hydrophobic side chains not involved in interhelical contacts, and (iii) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.

NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin

Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O2 binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both α- and β-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring 1H and 15N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.

Foldons, protein structural modules, and exons.

Foldons, which are kinetically competent, quasi-independently folding units of a protein, may be defined using energy landscape analysis. Foldons can be identified by maxima in a scan of the ratio of a contiguous segment's energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. The predicted foldons are compared with the exons and structural modules for 16 of the 30 proteins studied. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules, but there are marked sequence-dependent effects. There is only a weak correlation of foldons to exons. For gammaII-crystallin, myoglobin, barnase, alpha-lactalbumin, and cytochrome c the foldons and some noncontiguous clusters of foldons compare well with intermediates observed in experiment.

Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway.

Apomyoglobin folding proceeds through a molten globule intermediate (low-salt form; I1) that has been characterized by equilibrium (pH 4) and kinetic (pH 6) folding experiments. Of the eight alpha-helices in myoglobin, three (A, G, and H) are structured in I1, while the rest appear to be unfolded. Here we report on the structure and stability of a second intermediate, the trichloroacetate form of the molten globule intermediate (I2), which is induced either from the acid-unfolded protein or from I1 by > or = 5 mM sodium trichloroacetate. Circular dichroism measurements monitoring urea- and acid-induced unfolding indicate that I2 is more highly structured and more stable than I1. Although I2 exhibits properties closer to those of the native protein, one-dimensional NMR spectra show that it maintains the lack of fixed side-chain structure that is the hallmark of a molten globule. Amide proton exchange and 1H-15N two-dimensional NMR experiments are used to identify the source of the extra helicity observed in I2. The results reveal that the existing A, G, and H helices present in I1 have become more stable in I2 and that a fourth helix--the B helix--has been incorporated into the molten globule. Available evidence is consistent with I2 being an on-pathway intermediate. The data support the view that apomyoglobin folds in a sequential fashion through a single pathway populated by intermediates of increasing structure and stability.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.