Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution.

Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form alpha-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the alpha-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5 degrees C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis.

IR spectroscopy of isotope-labeled helical peptides: probing the effect of N-acetylation on helix stability.

The effect of N-acetylation on the conformation of alanine-rich helical peptides is examined using isotope-edited Fourier transform infrared (FTIR) spectroscopy. A series of peptides with sequence AA(AAKAA)(3)AAY has been prepared; each peptide incorporates four (13)C-labeled alanines. These peptides have two amide I' bands in their FTIR spectra: one corresponding to the (12)C amino acids, and one assigned to the (13)C amino acids. The intensity and frequency of the (13)C amide I' band varies systematically with the position of the labels in the sequence and the presence or absence of an N-acetyl capping group. The intensity of the (13)C amide I' band correlates with helix stability at the labeled residues as predicted by thermodynamic models of the helix-coil transition. These results suggest that FTIR spectroscopy combined with specific isotope labeling can be used to dissect the conformation of helical peptides at the residue level.

Hydrogen bonding modulates binding of exogenous ligands in a myoglobin proximal cavity mutant.

In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. (1)H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.

Trans effects in nitric oxide binding to myoglobin cavity mutant H93G.

When nitric oxide (NO) binds to heme proteins, it exerts a repulsive trans effect on the proximal ligand, resulting in weakening or rupture of the proximal ligand-iron bond. The general question of whether NO binding generates a five-coordinate complex with proximal ligand release is important for the function of enzymes such as guanylate cyclase. This question can be addressed by studying NO binding to the myoglobin cavity mutant H93G, where the proximal histidine has been replaced by glycine. When this protein is expressed in the presence of imidazole (Im), an imidazole molecule occupies the proximal cavity and serves as a ligand to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554]. This proximal imidazole can be exchanged for a variety of exogenous ligands [DePillis, G.D., Decatur, S. M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. While CO binds to H93G(Im) to form a stable six-coordinate complex similar to that of the wild type and NO binds to wild-type myoglobin to form a six-coordinate complex, we find that the binding of NO to H93G(Im) under similar conditions results in the cleavage of the exogenous imidazole-iron bond at neutral pH, leaving a five-coordinate heme-NO complex, H93G-NO, inside the protein. When a large excess of imidazole is added to this five-coordinate NO complex, a six-coordinate complex can be formed; thus, the binding constant of a sixth ligand to the five-coordinate H93G-NO complex can be measured. This is found to be several orders of magnitude smaller than the binding constant of Im to the carbonmonoxy, deoxy, or the metcyano forms of protein. By replacement of Im with methyl-substituted imidazoles which have hindered or strained binding conformations, this binding constant can be reduced further and some of the factors responsible for favoring the five-coordinate form can be elucidated. Thus, the cavity mutant H93G provides a novel model system for studying the factors that control the coordination state of NO complexes of heme proteins and serves as a bridge between synthetic heme model complexes in simple solvents and site-directed mutants in the structured environment found in proteins.

Modulation of protein function by exogenous ligands in protein cavities: CO binding to a myoglobin cavity mutant containing unnatural proximal ligands.

A variety of heterocyclic ligands can be exchanged into the proximal cavity of sperm whale myoglobin mutant H93G, providing a simple method for introduction of the equivalent of unnatural amino acid side chains into a functionally critical location in this protein. These modified proteins bind CO on the distal side. 1H NMR data on H93G(Im)CO, where Im is imidazole, demonstrate that the structure of the distal heme pocket in H93G(Im)CO is very similar to that of wild type; thus, the effects of the proximal ligand's properties on CO binding can be studied with minimal perturbation of distal pocket structure. The exogenous proximal ligands used in this study include imidazole (Im), 4-methylimidazole (4-MeIm), 4-bromoimidazole (4-BrIm), N-methylimidazole (N-MeIm), pyridine (Pyr), and 3-fluoropyridine (3-FPyr). Substitution of the proximal ligand is found to produce substantial changes in the CO on and off rates, the equilibrium binding constant, and the vibrational stretch frequency of CO. Many of the changes are as large as those reported for distal pocket mutants prepared by site-directed mutagenesis. The ability to systematically vary the nature of the proximal ligand is exploited to test the effects of particular properties of the proximal ligand on CO binding. For example, 4-MeIm and 4-BrIm are similar in size and shape but differ significantly in pKa. The same relationship is true for Pyr and 3-FPyr. By comparison of the IR spectra and CO recombination kinetics of these complexes, the effects of proximal ligand pKa on the CO binding are assessed. Likewise, N-MeIm and 4-MeIm are similar in size and pKa but differ in their ability to hydrogen bond to amino acid residues in the proximal cavity. Comparisons of IR spectra and CO binding kinetics in these complexes reveal that proximal ligand conformation and hydrogen bonding affect the kinetics of CO binding. The mechanism of proximal ligand exchange between solution and the proximal cavity in CO complexes was investigated by obtaining the 19F NMR spectrum of H93G(3-FPyr)CO, whose 19F signal can be observed without interference from resonances of the protein. The proximal ligand is found to exchange within a few seconds by saturation transfer. This exchange rate is about 2 orders of magniture faster than what is observed for the isoelectronic metcyano complex [Decatur, S. M., & Boxer, S. G. (1995) Biochemistry 34, 2122-2129]; in both the ferrous CO and ferric cyano complexes, the proximal ligand exchange rate is independent of ligand concentration. These results suggest that the rate-limiting step in proximal ligand exchange is breakage of the iron-ligand bond, followed by rapid diffusion of the ligand through the protein to bulk solution.

A test of the role of electrostatic interactions in determining the CO stretch frequency in carbonmonoxymyoglobin.

The vibrational frequency of CO bound to myoglobin can be varied by up to 60 cm-1 by making site-specific mutations in the distal pocket. These changes may result from specific chemical interactions between distal amino acids and the CO or from changes in the electrostatic field of the distal pocket. In this paper, we separate the relative contributions of these two effects by comparing the IR spectra of the carbonmonoxy complexes of human myoglobin mutants V68N, V68D, and V68E. The effect of replacing valine with these polar amino acids on the electrostatic environment of the distal heme pocket has been independently determined earlier by measurements of the heme reduction potential and electronic absorption spectral band shifts. While all three mutations result in a negative dipole pointing towards the CO ligand, the CO stretch frequency shifts differently in each case. These differences are attributed to specific chemical interactions between the amino acids and the CO ligand.

1H NMR characterization of myoglobins where exogenous ligands replace the proximal histidine.

The role of the proximal ligand in determining the structure and ligand binding properties of sperm whale myoglobin has been investigated using the mutant H93G(L), where the proximal histidine has been replaced with glycine, creating a cavity which can be occupied by a variety of exogenous ligands, L, to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554; DePillis, G.D., Decatur, S.M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. In this report, we present the assignments of selected protons of the heme and heme pocket residues in the metcyano complexes of H93G with Im and a series of methyl-substituted Ims [H93G(Im)CN, H93G(N-MeIm)CN, H93G(2-MeIm)CN, H93G-(4-MeIm)CN]. Each complex has a unique 1H NMR spectrum, providing a fingerprint for documenting the ligand exchange phenomenon. Moreover, the identification of NOEs between the protons of proximal ligands and protons of proximal pocket amino acid residues confirms that the new ligand occupies the proximal cavity in solution. The pattern of hyperfine-shifted heme methyl resonances in H93G(Im)CN is very different from that of wild-type Mb, consistent with the differences compared to wild-type in is very different from that of wild-type Mb, consistent with the differences compared to wild-type in orientation of the proximal imidazole observed in the X-ray crystal structure of H93G(Im) [Barrick, D. (1994) Biochemistry 33, 6546-6554]. Addition of deuterated Im to H93G(Im)CN permits direct observation of exchange of proximal ligands with ligands from solution; exchange of Im for deuterated Im in the metcyano complex occurs with half-life of around 10 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Anatomy and dynamics of a ligand-binding pathway in myoglobin: the roles of residues 45, 60, 64, and 68.

In order for diatomic ligands to enter and exit myoglobin, there must be substantial displacements of amino acid side chains from their positions in the static X-ray structure. One pathway, involving Arg/Lys45, His64, and Val68, has been studied in greatest detail. In an earlier study (Lambright et al., 1989) we reported the surprising result that mutation of the surface residue Lys45 to arginine lowers the inner barrier to CO rebinding. Until then, it had been thought that this barrier primarily involves interior distal pocket residues such as His64 and Val68. In this report, we present a detailed study of the CO rebinding kinetics in aqueous solution of a series of single- and double-site mutants of human myoglobin at positions 64, 68, 45, and 60. On the basis of the observed kinetics, we propose that the effect of surface residue 45 on the inner barrier can be explained by a chain of interactions between surface and pocket residues. Very large, and in some cases unexpected, changes are observed in the kinetics of recombination and in the partitioning between geminate and bimolecular recombination.