Solution (1)H NMR study of the influence of distal hydrogen bonding and N terminus acetylation on the active site electronic and molecular structure of Aplysia limacina cyanomet myoglobin.

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution (1)H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small approximately 2 degrees rotation of the axial His in rWT relative to true WT and a approximately 3 degrees rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.

Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa.

The unfolding of the small cytochrome c551 from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c551 is found to be smaller than that of the larger horse cytochrome c (deltaGw = -8.2 vs. -9.7 kcal/mol); we propose that this finding is related to the absence of an 'omega' loop in the bacterial cytochrome. Cytochrome c551 loses most of its secondary structure at pH 1.5. The acid transition (pKA approximately 2) is highly cooperative (n > or =2); analysis of optical titrations and contact map suggests that (at least) His-16 (proximal Fe3+ ligand) and Glu-70 are both involved in the acid transition. The role of selected hydrophobic, electrostatic and conformational contributions to the overall stability has been investigated by protein engineering. The equilibrium characterization of wild-type and mutant cytochrome c551 supports the view that this small cytochrome is an interesting protein to analyze the thermodynamics and the kinetics of folding in comparison with the widely studied horse cytochrome c.

Unfolding of apomyoglobin from Aplysia limacina: the effect of salt and pH on the cooperativity of folding.

The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% alpha-helical under low salt conditions (2 mM citrate, 4 degrees C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F<-->I<-->U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (delta Gw(app) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in delta G(w(app)) occur without changes in n(app). The change in free energy of folding of I<-->F (delta Gw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. delta Gw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n(app) although there is no change in the alpha-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.

A myoglobin mutant designed to mimic the oxygen-avid Ascaris suum hemoglobin: elucidation of the distal hydrogen bonding network by solution NMR.

The solution 1H NMR structure of the active site and ligand dissociation rate for the cyanomet complex have been determined for a sperm whale myoglobin triple mutant Leu29(B10)-->Tyr, His64(E7)-->Gln, Thr67(E10)-->Arg that mimics the distal residue configuration of the oxygen-avid hemoglobin from Ascaris suum. A double mutant that retains Leu29(B10) was similarly investigated. Two-dimensional NMR analysis of the iron-induced dipolar shifts, together with the conserved proximal side structure for the two mutants, allowed the determination of the orientations of the paramagnetic susceptibility tensor for each complex. The resulting magnetic axes, together with paramagnetic relaxation and steady-state NOEs, led to a quantitative description of the distal residue orientations. The distal Tyr29(B10) in the triple mutant provides a strong hydrogen bond to the bound cyanide comparable to that provided by His64(E7) in wild-type myoglobin. The distal Gln64(E7) in the triple mutant is sufficiently close to the bound cyanide to severe as a hydrogen bond donor, but the angle is not consistent with a strong hydrogen bond. Dipolar contacts between the Arg67(E10) guanidinium group and the Gln64(E7) side chain in both mutants support a hydrogen-bond to the Gln64(E7) carbonyl group. The much lower oxygen affinity of this triple mutant relative to that of Ascaris hemoglobin is concluded to arise from side-chain orientations that do not allow hydrogen bonds between the Gln64(E7) side-chain NHs and both the ligand and Tyr29(B10) hydroxyl oxygen. Cyanide dissociation rates for the reduced cyanide complexes are virtually unaffected by the mutations and are consistent with a model of the rate-determining step as the intrinsically slow Fe-C bond breaking that is largely independent of any hydrogen bonds to the cyanide nitrogen.

Interactions among residues CD3, E7, E10, and E11 in myoglobins: attempts to simulate the ligand-binding properties of Aplysia myoglobin.

Site-directed mutations have been introduced singly and in combination at residues lysine/arginine45 (CD3), histidine64 (E7), threonine67 (E10), and valine68 (E11) in pig and sperm whale myoglobins. The mutations probe the roles of these key distal pocket residues and represent attempts to mimic the heme environment of Aplysia limacina myoglobin which achieves moderately high O2 affinity in the absence of a distal histidine. In the mollusc myoglobin, arginine-E10 is believed to swing into the heme pocket and provide a hydrogen bond to the bound O2. The association and dissociation rate constants for oxygen and carbon monoxide binding to H64V, T67A, T67V, T67E, T67R, V68I, V68T, H64V-T67R, H64V-V68T, H64V-V68I, and H64V-T67R-V68I pig myoglobin mutants and T67R, H64V-T67R, and R45D-H64V-T67R mutants of sperm whale myoglobin have been measured using stopped-flow rapid mixing and flash photolysis techniques. Replacement of histidine-E7 with valine in either pig or sperm whale myoglobin drastically lowers O2 affinity while increasing CO affinity. Two second-site mutations, T67R and V68T, increase O2 affinity in the H64V mutant, even though when introduced singly these mutations have no effect or lower KO2, respectively. However, the oxygen affinities of the H64V-T67R mutants are 5-10-fold lower than that of A. limacina myoglobin. The crystal structure of the pig H64V-T67R double mutant reveals that the valine-E7 side chain is approximately 1 A closer to the heme plane than in the mollusc protein which may restrict access of the arginine-E10 side chain into the heme pocket. The O2 affinity of the H64V-T67R double mutant is not altered by the R45D replacement but is reduced 10-fold by the V68I mutation. The interactive effects of the T67R, V68I, and V68T mutations with the H64V substitution are discussed in terms of O2, CO, and N3-binding and the crystal structures of the H64V-T67R, H64V-V68I, and H64V-V68T double-mutant proteins. In many instances, the effects of second-site mutations in the valine64 background are the opposite of those observed for the corresponding single mutations in the wild type background. These results can be understood in terms of the changes in the rate-determining steps for ligand association and dissociation and the loss of distal pocket water molecules which follow replacement of histidine64 by valine.

Formate binding to ferric wild type and mutant myoglobins thermodynamic and X-ray crystallographic study.

The X-ray crystal structure of the formate derivative of ferric loggerhead sea turtle (Caretta caretta) Mb has been determined at 2.0 A resolution (R = 0.164) by difference Fourier techniques. Formate, sitting in the central part of the heme distal site, is coordinated to the heme iron as unidentate ligand, through the O1 oxygen atom, and is hydrogen bonded to the distal His64(E7) NE2 atom through O2. Thermodynamics for formate binding to ferric loggerhead sea turtle Mb, sperm whale Mb, Aplysia limacina Mb, as well as to the VR and VRS mutants of sperm whale Mb were obtained between pH 4.5 and 8.5, at 20.0 degrees C. These results, representing the first structure of a ferric hemoprotein:formate complex solved by X-ray crystallography, outline the role of amino acid residues at positions E7, F8 and E10 in modulating ligand binding properties of oxygen carrying proteins.

Structural factors governing azide and cyanide binding to mammalian metmyoglobins.

The structural factors governing azide and cyanide binding have been examined by measuring the effects of 46 mutations at key topological positions in the distal pocket in sperm whale, pig, and human myoglobin. Replacement of His64 (E7) with smaller amino acids results in dramatic increases in the association rate constant for azide binding primarily due to relief of steric hindrance imposed by the imidazole side chain. Gln64 and His64 (native) metmyoglobins have abnormally low rate constants for azide dissociation (0.1-0.3 s-1) due to direct hydrogen bonding between the N epsilon atoms of these residues and the bound ligand. Mutations at positions 67(E10) and 68(E11) produce large but complex changes in the azide binding parameters as a result of both steric and electrostatic effects, which alter water coordination, influence the rate of anion movement into the distal pocket, and affect the stability of the Fe-N3 bond. Replacement of Phe46 with Leu or Val and substitution of Arg(Lys)45 with Glu and Ser cause disorder in the position of the distal histidine side chain and result in 4-700-fold increases in both k'N3 and kN3 but produce little change in overall azide affinity. All of these results suggest strongly that azide enters the distal pocket of native myoglobin through a polar channel that is regulated by a His64 "gate." In contrast to azide binding, the rate constant for cyanide association decreases 4-300-fold when the distal histidine is replaced with apolar residues. His64, Gln64, and distal pocket water molecules appear to facilitate deprotonation of HCN, which is the major kinetic barrier to cyanide binding at neutral pH.

A new point mutation of the prion protein gene in Creutzfeldt-Jakob disease.

Complete sequencing of the prion protein open reading frame of a 68-year-old woman affected by a familial form of Creutzfeldt-Jakob disease (CJD) revealed a new mutation at codon 210 resulting in the substitution of isoleucine for valine. Moreover, a new 24-bp deletion encompassing codons 54 to 61 or 62 to 69 was found in the other allele. Four of the 17 asymptomatic relatives tested carry the 210 mutation. Two of them were 81 and 82 years old. Four of 22 patients with CJD whose recorded familial history was negative for demented illnesses, but none of 103 healthy control subjects, tested positive for the 210 mutation. These data suggest that the 210 mutation is associated with CJD, but that environmental factors or incomplete penetrance may contribute to the development of the disease. This finding also suggests that in Italy, familial CJD is more common than previously reported.

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.

Structural and functional characterization of sperm whale myoglobin mutants: role of arginine (E10) in ligand stabilization.

1H NMR and ligand-binding data were used to assess the role of residue Arg(E10) in ligand stabilization of several site-directed mutants, all carrying the His(E7) to Val substitution, obtained using a synthetic sperm whale myoglobin gene. Arg(E10) was previously found to form a hydrogen bond with the ligand in fluoro-, azido- and cyanomet derivatives of Aplysia limacina myoglobin, which lacks the distal His(E7) [Qin, J., La Mar, G. N., Ascoli, F., Bolognesi, M., & Brunori, M. (1992) J. Mol. Biol. 224, 891-897]. NMR analysis of the paramagnetically induced relaxation, hyperfine shift patterns, and dipolar connectivities shows that Arg(E10) also falls into the distal pocket in the engineered sperm whale myoglobin mutants and resides at an H-bonding distance from the Fe(3+)-bound cyanide. The rate constant for cyanide dissociation from the ferrous derivative was determined by stopped-flow experiments; the ligand stabilization achieved by Arg(E10) is similar to that exerted by His(E7) in wild-type sperm whale myoglobin, and both are very different from the His(E7)Val single mutant. Contrary to that for the wild-type, the cyanide dissociation rate constant for the mutant containing Arg(E10) is essentially independent of pH (from 6 to 9), as expected on the basis of the guanidinium group of Arg having a pK > 10. This finding is consistent with the NMR data in which the chemical shift of the Arg(E10) N epsilon H is insensitive to pH (6-9), as is also observed in Aphysia limacina cyanometmyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of a distal site double mutant of sperm whale myoglobin at 1.6 A resolution.

The three-dimensional structure of sperm whale myoglobin His64(E7)-->Val,Thr67(E10)-->Arg double mutant has been studied by X-ray crystallography at 1.6 A resolution, and refined to a crystallographic R-factor of 0.197. The Arg67(E10) side chain is extended in the direction of the ligand binding site, and its NH1 atom is at a distance of 3.11 A from the NH1 atom of Arg45(CD3), which is also pointing towards the distal site. Both are kept in this position by hydrogen bonding and electrostatic interactions with a solvent sulfate ion, located amongst the two, on the protein surface. No liganded water molecule is present at the sixth coordination position of the Fe(III) heme.

Control and recognition of anionic ligands in myoglobin.

Equilibrium and kinetic experiments on site-directed mutants of a synthetic sperm whale myoglobin (Mb) gene have been performed. Results on the reactivity on both ferric and ferrous wild type and mutants Mb's are presented. Analysis of ligand binding to His (E7) Val and His (E7) Val-Thr (E10) Arg mutants compared to wild-type sperm whale, horse and Aplysia limacina Mb's, shows that the introduction of an arginyl residue at the topological position E10 greatly enhances the stability of the various Mg:heme ligand adducts. Alternative mechanisms of ligand stabilization may therefore be operative in Mb's lacking the distal histidine.