Structure Of Sperm Whale Myoglobin Research Articles

ЭЛЕКТРОСТАТИЧЕСКОЕ ВЗАИМОДЕЙСТВИЕ ГЛОБИНОВ С ФОСФОЛИПИДНЫМИ МЕМБРАНАМИ

The present study aimed to analyze the role of electrostatic interactions contributing to the stability of the native conformations of the heme group in the structure of sperm whale myoglobin (SWMb), horse heart myoglobin (HHMb), hemoglobin I (HbI) from the botfly Gasterophilus intestinalis (giHbI) and monomeric and dimeric hemoglobins HbI and HbII from the mollusk Lucina pectinata (lpHbI and lpHbII) as well as investigate a possible reason of destabilization due to interaction with negatively charged phospholipid membranes. It was shown that the native conformation of the heme cavity in these globins, both in its proximal and distal sides, is sustained by a system of hydrogen bonds involving the proximal and distal protein residues, both heme propionic acid groups and the nearby polar amino acids on the protein surface (His, Arg, Lys). The hydrogen bond network in the proximal part of the heme pocket controls the position of the Fe atom outside or in the protoporphyrin plane, affecting the efficiency of ligand binding, while in the distal part of the heme cavity the hydrogen bond network formed with the participation of the distal protein residue (HisE7 in swMb, hhMb and giHbI, and GlnE7 in lpHbII) should, apparently, stabilize conformation where the protein is able to donate hydrogen to O2 ligand...

Thermal unfolding of myoglobin in the Landau-Ginzburg-Wilson approach.

The Landau-Ginzburg-Wilson paradigm is applied to model the low-temperature crystallographic Cα backbone structure of sperm whale myoglobin. The Glauber protocol is employed to simulate its response to an increase in ambient temperature. The myoglobin is found to unfold from its native state by a succession of α-helical intermediates, fully in line with the observed folding and unfolding patterns in denaturation experiments. In particular, a molten globule intermediate is identified with experimentally correct attributes. A detailed, experimentally testable contact map is constructed to characterize the specifics of the unfolding pathway, including the formation of long-range interactions. The results reveal how the unfolding process of a protein is driven by the interplay between, and a successive melting of, its modular secondary structure components.

Determination of Ligand Pathways in Globins: APOLAR TUNNELS VERSUS POLAR GATES

Although molecular dynamics simulations suggest multiple interior pathways for O(2) entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.

Computer Simulations of the Refolding of Sperm Whale Apomyoglobin from High-Temperature Denaturated State

The refolding mechanism of apomyoglobin (apoMb) subsequent to high-temperature unfolding has been examined using computer simulations with atomic level detail. The folding of this protein has been extensively studied experimentally, providing a large database of folding parameters which can be probed using simulations. In the present study, 4-folding trajectories of apoMb were computed starting from coiled structures. A crystal structure of sperm whale myoglobin taken from the Protein Data Bank was used to construct the final native conformation by removal of the heme group followed by energy optimization. The initial unfolded conformations were obtained from high-temperature molecular dynamics simulations. Room-temperature refolding trajectories at neutral pH were obtained using the stochastic difference equation in length algorithm. The folding trajectories were compared with experimental results and two previous molecular dynamics studies at low pH. In contrast to the previous simulations, an extended intermediate with large helical content was not observed. In the present study, a structural collapse occurs without formation of helices or native contacts. Once the protein structure is more compact (radius of gyration<18 A) secondary and tertiary structures appear. These results suggest that apoMb follows a different folding pathway after high-temperature denaturation.

Structural characterization of non-native states of sperm whale myoglobin in aqueous ethanol or 2,2,2-trifluoroethanol media

The effects of aqueous ethanol or 2,2,2-trifluoroethanol media on the structure of sperm whale myoglobin have been investigated by absorption, CD, and NMR spectra. The structural properties of myoglobin such as heme environments, helix contents, protein folding, and interactions between heme and the protein moiety have been sharply manifested in these spectra. The characterization demonstrated that alcohol-induced conformational change of myoglobin depends on the nature of alcohol and its concentration. It was shown for the first time that, upon the alcohol-induced denaturation of myoglobin, heme is released from partially denatured protein of which helix contents is altered by only about 20% relative to that of native state. Myoglobin has shown to unfold and refold reversibly by controlling the alcohol concentration. Novel methods for the preparation of apomyoglobin and in situ reconstitution of apomyoglobin with heme, based on the alcohol-induced denaturation of the protein, were presented.

Myoglobin, a paradigm in the study of protein dynamics.

More than 40 years have passed since Kendrew determined the structure of sperm whale myoglobin. It gave us the first insight into the impressive architecture of an alpha helical protein folded via loops into a well ordered, nearly spherical molecule. As time progressed, thousands of new protein structures were published with increasing resolution. The myoglobin structure was also continuously improved and, three years ago, a resolution of 0.9 A was achieved. A wide variety of spectroscopic techniques have been applied to study the dynamics of this molecule and the electronic properties of the heme iron atom. Even nowadays, experiments on myoglobin are still of great interest for a number of reasons: Physical research cannot go into all details in a large number of systems. In semiconductor physics, most concepts were developed by investigating germanium and silicon and were subsequently used to understand more complicated semiconductor systems; myoglobin plays a similar role in molecular biophysics. Concepts like "energy landscape", "conformational substates", "dynamic transition", or "protein-specific motions" should be of general relevance for understanding the physics of biological macromolecules. In biology, functionally important processes often occur at conditions and time scales where physical experiments are extremely difficult and yield ambiguous results. Going to low temperatures often allows one to separate the different dynamic processes. Another helpful tool is molecular engineering. Specific alterations in the sequence of the protein open the possibility to modify reaction rates and to retard the kinetics.

Probing Substates in Sperm Whale Myoglobin Using High-Pressure Crystallography

Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.

Structural and functional properties of designed globins

De novo design of artificial proteins is an essential approach to elucidate the principles of protein architecture and to understand specific functions of natural proteins and also to yield novel molecules for medical and industrial aims. We have designed artificial sequences of 153 amino acids to fit the main-chain framework of the sperm whale myoglobin structure based on the knowledge-based energy functions to evaluate the compatibility between protein tertiary structures and amino acid sequences. The synthesized artificial globins bind a single heme per protein molecule as designed, which show well-defined electrochemical and spectroscopic features characteristic of proteins with a low-spin heme. Redox and ligand binding reactions of the artificial heme proteins were investigated and these heme-related functions were found to vary with their structural uniqueness. Relationships between the structural and functional properties are discussed.

Design and synthesis of a globin fold.

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.

Electrostatic multipole representation of a polypeptide chain: An algorithm for simulation of polypeptide properties

A method for describing a polypeptide chain based on an electrostatic multipole representation is introduced. The main features of the description are outlined. Appropriate energy functions for nonbonded interactions are developed. The full atomic representation may be retrieved from the electrostatic multipole representation at any point in a calculation. The multipole description and the energy functions are tested by calculation of steric maps for different amino acid side-chain groups. The ability to calculate energetically stable structures is demonstrated by energy conformation maps and the results of energy calculations in optimal secondary structural elements. Results from dynamics simulations of helical chains of polyglycine, polyalanine, polyvaline, and a 21-residue helix obtained from the crystal structure of sperm whale myoglobin are included to demonstrate the efficiency of the algorithm. It is demonstrated that this description of the polypeptide chain is both simple and complete and will allow for the rapid simulation of chain dynamics without loss of essential information about the chain. © 1996 by John Wiley & Sons, Inc.

Basi molecolari del riconoscimento eme:leganti nella mioglobina di capodoglio (Physeter catodon) e diAplysia limacine

The crystal structure of sperm whale myoglobin (Mb):azide complex has been determined and refined at 1.8 A resolution. The ligand is coordinated to the heme iron, and deeply buried in the inner part of the distal site. The structural organization of this complex differs substantially from that observed inA. limacina Mb: azide complex, in which the iron coordinated ligand is directed towards the outer solvent region of the protein. In the case of sperm whale Mb the bound ligand is stabilized by a hydrogen bond to residue HisE7; in the case ofA. limacina Mb the ligand is directly hydrogen bonded to ArgE10. The structures of the complexes described underline different mechanisms of ligand recognition and stabilization in the two Mb’s, and allow a rational interpretation of the different kinetic and thermodynamic properties observed in globins lacking a polar residue at the distal E7 site.

X-ray crystal structure of a recombinant human myoglobin mutant at 2·8 Å resolution

We have grown crystals in trigonal space group P3(2)21 of a mutant human myoglobin, aquomet form, in which lysine at position 45 has been replaced by arginine and cysteine at position 110 has been replaced by alanine. Suitable crystals of native recombinant human myoglobin have not been obtained. We have used the molecular replacement method to determine the X-ray crystal structure of the mutant at 2.8 A resolution. At the present stage of refinement, the crystallographic R-value for the model, with tightly restrained stereochemistry, is 0.158 for 5.0 to 2.8 A data. As expected, the overall structure is quite similar to the sperm whale myoglobin structure. Arginine 45 adopts a well-ordered conformation similar to that found in aquomet sperm whale myoglobin.

A structure of sperm whale myoglobin at a nitrogen gas pressure of 145 atmospheres.

A structure of sperm whale metmyoglobin under a nitrogen gas pressure of 145 atm (2200 psi) has been solved by X-ray diffraction using data to 2.0-A resolution. The perturbation of the gas pressure on the overall structure of the protein is minimal with a root mean square deviation of backbone atoms between the pressurized and unpressurized structures of 0.22 A. Additional electron density is observed, however, in two cavities of the protein molecule. The density is interpreted as a nitrogen molecule bound in the proximal cavity and as a water molecule hydrogen bonded in a separate cavity (cavity 3). In addition, alternate conformations are observed for three internal residues (Leu-135, Phe-138, and Ile-142) that border these cavities. These alternate conformations are not observed in atmospheric pressure structures and are presumed due to the effects of pressure and/or gas binding. The appearance of these alternate conformations implies a repacking of the protein interior and produces a new distribution of cavity spaces. The profile of the Debye-Waller factors for the pressurized structure is similar to that for the room pressure except for a small increase in the distal region (residues 61-69) of the protein.

Thermodynamics of carp metmyoglobin unfolding

1. 1. The conformational free energy of carp lateral muscle metmyoglobin at 25°C pH 8 is 9.0 ± 0.5 kcal/mol as estimated from isothermal unfolding by both urea and guanidinium chloride. 2. 2. A novel procedure for the simultaneous analysis of acid and guanidinium chloride unfolding data is described. 3. 3. Acid denaturation data suggest that binding of five protons to histidyl residues occurs on unfolding. 4. 4. Correlation of sequences and conformational stabilities of several myoglobins according to the tertiary structure of sperm whale myoglobin suggests an evolutionary turnover of side chain-side chain interactions.

A simple method for the calculation of low energy packings of α-helices—A threshold approximation. I. The use of the method to estimate the effects of amino acid substitutions, deletions and insertions in globins

A simple method is developed for computing low energy packings in alpha-helical proteins. This method is based on a simplified representation of a protein molecule in which the backbone of the alpha-helix is represented by a cylinder and the side chains of the amino acids by hard spheres. The energy function includes hydrophobic, electrostatic and Van der Waals' interactions. This method is used to compute low energy packings of an assembly of the alpha-helices of a globin molecule. The effect of mutations, deletions and insertions of a single amino acid resulting in drastic distortion of the spatial structure of sperm whale myoglobin was studied. The results demonstrated that these events can produce a breakdown of the spatial structure of the protein molecule.

The effects of mutations, deletions and insertions of single amino acids on the three-dimensional structure of globins

Using the method of calculation of low energy packings of α-helices, we studied the effects of mutations, deletions and insertions of amino acids on the structure of sperm whale myoglobin. It was found that these events can cause complete breakdown of this structure.

Semisynthesis of sperm whale myoglobin by fragment condensation.

Reconstruction of the sperm whale myoglobin structure was accomplished by a series of aqueous condensations of suitably protected synthetic myoglobin fragments to a large fragment prepared from the native protein. Reaction of N alpha,N epsilon 19-acetimidomyoglobin with 3-bromo-2-(2-nitrophenylsulfenyl)skatole (BNPS-skatole) yielded the fragment corresponding to residues 15-153. The covalent structure was reformed by sequential coupling of the N-hydroxysuccinimide esters of o-nitrophenylsulfenyl-L-tryptophan (residue 14) and selectively protected peptides corresponding to residues 1-5 and 6-13, which were synthesized by the solid-phase method and removed from the resin by methoxide-catalyzed methanolysis. A mixed aqueous solvent system containing methanol and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine/trifluoroacetic acid buffer (Quadrol) solubilized the peptide and protein fragments during the condensations. Replacement of the heme moiety and immunoaffinity chromatography made possible the isolation and purification of the reconstructed native molecule. The development of this nondestructive synthetic procedure allows investigation of the structural and functional significance of individual residues by isotopic enrichment or selective amino acid substitutions.

Antigenic structure of sperm whale myoglobin. II. Characterization of antibodies preferentially reactive with peptides arising in response to immunization with the native protein.

The antigenicity of peptide fragments derived from CNBr-cleaved sperm whale Mb has been tested in competitive inhibition assays. These peptides appeared to be very poor inhibitors in a reaction involving radiolabeled sperm whale Mb with antipeptide antibodies, requiring about 10(4)-fold excess of peptides to achieve the same level of inhibition as cold native Mb. However, cold peptides competed much better with radiolabeled peptides for antibody binding than the native protein. These antibodies, preferentially reactive with peptides, originated from sera of animals immunized with the native molecule. This indicates heterogeneity of the antibodies produced against Mb and suggests the presence of diverse antibody subpopulations reactive with different antigenic forms of the protein. Results presented in this paper illustrate that competitive radioimmunoassays preferentially measure antibodies to the radiolabeled species, and this phenomenon can be used to demonstrate distinct populations among antibodies isolated to a single peptide.

Antigenic structure of sperm whale myoglobin. I. Partition of specificities between antibodies reactive with peptides and native protein.

We have resolved anti-sperm whale Mb antibodies into two distinct populations: One population is reactive only with the native molecule, and the second is reaction with both Mb and peptide fragments. CNBr cleavage of sperm whale Mb yields three peptides: peptide I (1-55), peptide II (56-131), and peptide III (132-153). Immunoadsorbent columns made with these three peptides were used to fractionate antibody to sperm whale Mb. These columns cumulatively bind 60 to 70% of the total antibody present in the four sera studied (the amount of antibody bound by each peptide was proportional to its size). The remaining 30 to 40% of antibody could only bind specifically to an immunoadsorbent column of native Mb. This result indicates that not all antigenic determinants on the native Mb molecule are represented on peptide fragments.

Dissection of the molecular parameters for T-cell recognition of a myoglobin antigenic site.

Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm-whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T-cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145-153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time up to 22 residues in length (peptide 132-153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr-151 and Ala-144. The T-cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb-primed mice afforded T-cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145-153 and 143-153) and more so to the longer peptides 135-153 and 132-153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity of T-cells. No response was obtained also to intermediate-sized peptides. The latter result, due to an unfavorable mode of folding, suggested a conformational dependency in T-lymphocyte recognition and was confirmed by additional studies employing peptide priming. Thus priming with either the 11- or 22-residue peptides afforded T-cells which proliferated to both of these two peptides, but not to intermediate-sized peptides. More importantly, peptide-primed cells did not proliferate on challenge in vitro with Mb. This indicated that the in vivo recognition (priming) was to the unfolded peptide and these cells will not therefore recognize native Mb in vitro. Tolerization of neonatal mice with native Mb, which was total for the native protein at the T-cell level, had no effect on in vivo recognition (priming) and in vitro proliferation due to the unfolded peptide. Conversely, tolerization by any unfolded peptide had little or no effect on in vivo recognition of, and proliferation to, native Mb. It was concluded that T-cell recognition of native proteins (or at least of Mb) is dependent on protein conformation. A peptide may not cause proliferation of native protein-primed T-cells even though it contains an antigenic site. Finally, macrophage presentation of protein antigens is not related to processing via fragmentation of antigen by macrophage.