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Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

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DEVKUMAR MUSTAFI AND MARVIN W. MAKINEN

nitroxyl spin-labels, the or component of the tensor is critical for selection of molecular orientation. In the case of setting to the –7/2 parallel component of the EPR spectrum (cf., Fig. 1) selects those molecules for which the V = O bond or component of the tensor is parallel to the laboratory magnetic field, i.e., the equatorial x,y-plane is perpendicular to Similarly for spin-labels, setting to the low-field component of the EPR spectrum (cf., Fig. 2) selects those molecules for which the component of the tensor is parallel to i.e., the molecular x,y-plane is similarly perpendicular to Correspondingly, setting to the –3/2 perpendicular component of the EPR spectrum of (cf., Fig. 1) selects molecules for which the x,y-molecular plane is parallel to the laboratory field. In the case of nitroxyl spin-labels, the central prominent absorption feature arises from molecules of all orientations, including those for which the molecular plane is perpendicular to

Figs. 3 and 4 compare the expected pattern of observed ENDOR splittings as a function of the positions of protons and the orientation of with respect to of or with respect to of spin-labels, respectively. When is perpendicular to the molecular plane, of a proton located along the symmetry axis or of a proton located near or in the x,y-plane will be observed. On the other hand, if is parallel to the x,y-plane, of a proton located along the symmetry axis and both and of a proton located near or in the x,y-plane are observed. On this basis, the ENDOR splittings for both types of paramagnetic probes can be classified into three categories:

Figure 3. Schematic illustration of the relationships of the symmetry axis of the ion to the principal axes of the g tensor and of the hf tensors of nearby protons. Left diagram: Direction of the molecular axes with respect to the V=O bond. Central diagram: Relative positions of protons near the molecular x,y-plane and near or along the z-axis. Each circle represents an orientation of within the g-axis system. Right diagram: Principal hfc components that are detected for equatorially or axially positioned protons according to whether is aligned parallel or perpendicular to the z-axis.

ANGLE-SELECTED ENDOR

101

Figure 4._Magnetic interactions governing angle-selected ENDOR in a nitroxyl spin-label. Upper left-hand portion: Relationships of molecular axes to the principal axes ofthe tensor of the nitroxyl group and of the A tensors of nearby protons located in the x,y-plane or on the z axis. Lower left-hand portion: Frozen solution EPR spectrum of a nitroxyl spin-label indicating settings (A, B) for microwave saturation. (cf., Fig. 2). Right-hand portion: ENDOR spectra of N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carbonyl)-L-alaninate enriched with deuterium except at recorded for settings A and B of the static magnetic field The abscissa measures the observed ENDOR shift (observed frequency of resonance minus the proton Larmor frequency). Reprinted from Mustafi et al. (2001) with permission.

i.splittings that are observed only for the parallel orientation parallel to the or axis);

ii.splittings that are observed only for the perpendicular orientation

perpendicular to and

iii. splittings that are observed as common to both parallel and perpendicular orientations.

When g-anisotropy is small compared to the average g value, the maximum hf interaction energy occurs for a field oriented along the

electron-nucleus vector

and the minimum occurs when the field is

in the

For a proton in the x,y-plane, well resolved features corre-

sponding to

are observed which persist with an essentially constant

ENDOR shift at all

values (Hurst et al., 1985). This observation is an

important diagnostic feature since

the observed

couplings reach a

maximum splitting dependent on

(Yim and Makinen, 1986; Mustafi et

al., 1990b). Both

and

are observed when the magnetic field orientation

is near or in the molecular x,y-plane. This relationship provides the basis to analyze hf interactions in terms of nuclear coordinates for both types of paramagnetic species. For and nitroxyl spin-labels, we have observed hitherto only axially symmetric hf interactions such that each class of

protons gives rise to only

and

couplings. Furthermore, the principal

axes of the hf and

tensors

are observed to be coincident. These

102

DEVKUMAR MUSTAFI AND MARVIN W. MAKINEN

relationships may not always obtain. Surprisingly, in the case of in frozen glassy solutions, the covalent protons of the acetate ligand exhibit axially symmetric hf couplings while the protons of innershell coordinated water molecules within the same complex do not (Yim and Makinen, 1986). Similar observations have been made by others (Gochev and Yordanov, 1993; Zdravkova and Yordanov, 1994).

3.ENDOR CHARACTERIZATION OF STRUCTURED SOLVENT IN SMALL MOLECULE COMPLEXES AND IN PROTEINS

3.1 as a Structural Probe of and Sites in Proteins and Nucleotides

The molecular structure of was first elucidated through single crystal ENDOR studies (Atherton and Shackleton, 1980, 1984). However, results of early magnetic resonance studies supported the intuitive expectation that the penta-aquo vanadyl cation in solution would exhibit square pyramidal geometry with tetragonal symmetry, as had already been shown by X-ray for bis(acetylacetonato)oxovanadium(IV) (Dodge et al., 1961). The results of early nuclear magnetic resonance (NMR) studies using water were consistent with a square pyramidal complex; however, the presence of the fifth axial ligand could not be unambiguously established (Wuthrich and Connick, 1968). Albanese and Chasteen (1978) analyzed the EPR spectrum of in frozen aqueous medium and were the first to show quantitatively that the dipolar broadening produced by protons of inner sphere coordinated water molecules were consistent with as a complex of square pyramidal geometry, using vanadium-oxygen bond distances and valence angles determined crystallographically (Ballhausen et al., 1968). From proton ENDOR spectra of incorporated into host single crystals of in which the ion replaced plus one water molecule, Atherton and Shackleton (1980, 1984) determined the principal hfc components for all ten protons of the species. They showed that the traceless components of the principal hf tensors are nearly axially symmetric, expected for point-dipole interactions. Because the anisotropy of the various magnetic interactions was explored by rotation about crystal axes with respect to the applied magnetic field coordinates of each proton in the crystal could be assigned.

A more extensive study of the solvation structure of the ion was made by ENDOR spectroscopy of frozen solutions of in methanol and

ANGLE-SELECTED ENDOR

 

103

water-methanol mixtures on the basis of both and

ENDOR (Mustafi

and Makinen, 1988). In this study the structure of solvated

was

assigned through molecular modeling constrained by ENDOR-determined electron-nucleus distances. Although resonance features arising separately from methyl and hydroxyl protons overlap under these conditions, a total of seven pairs of resonance features due to hydroxyl protons with five pairs due to methyl protons were identified by selective deuteration. The principal hfc components for each class of protons were identified from ENDOR spectra with settings at the –7/2 parallel and –3/2 perpendicular EPR absorption features (cf., Fig. 1), corresponding to magnetic field orientations parallel and perpendicular to the V=O bond, respectively.

For each class of protons, the principal hfc components for the complex were axially symmetric. In Table 2 are listed the dipolar and isotropic hfc components, the metal-nucleus distances estimated

according to Eq. (4), and brief comments for each class of nuclei to indicate

the structural relationships of the ligand to

the

ion. Because the

unpaired electron is localized to the metal

orbital (Ballhausen and Gray,

1962), the isotropic contributions of ligand nuclei in the equatorial plane are significantly higher than for the axially coordinated ligand. Nevertheless, the values of from this study correspond very closely to values determined on the basis of single crystal ENDOR studies (Atherton and

Shackleton, 1980).

 

Fig. 5 illustrates the solvation structure of

with ENDOR

assigned innerand outer-sphere coordinated methanol molecules (Mustafi and Makinen, 1988). In this structure, the outer-sphere coordinated molecules were assigned orientations that could account for plausible hydrogen-bonding interactions with inner-sphere coordinated methanol molecules but which were compatible with the ENDOR-determined vanadium-nucleus distances in axial or equatorial oositions. It was shown by analysis of ENDOR spectra that was a unique complex formed only in neat methanol. In water-methanol mixtures, two types of species were identified: one with axially coordinated water trans to the vanadyl oxygen and the other with axially coordinated methanol. Both types of complexes were shown to have only equatorially coordinated water

molecules. The coordination geometry of

in neat methanol

and of

and of

in water-methanol

cosolvent mixtures was best accounted for as square-pyramidal with tetragonal symmetry. The structural detail obtained in this ENDOR study approached the precision associated with small molecule X-ray crystallographic studies.

104

DEVKUMAR MUSTAFI AND MARVIN W. MAKINEN

As shown in Fig. 1, inhomogeneous broadening observed in the spectrum of provides a particularly pertinent example of the difficulty to assign coordination environment on the basis of EPR alone. Axial and equatorial ligands do not make equivalent contributions to the shf broadening. In the case of Albanese and Chasteen (1978) were the first to point out that shf broadening by protons of axial ligands is weak compared to that of equatorial ligands. We have, furthermore, observed that the shf contributions of axial ligands can be masked by equatorial ligands. For instance, the line widths of EPR spectra of nucleotide complexes of formed with ADP (Mustafi et al., 1992) or 5'- GMP (Jiang and Makinen, 1995) and having composition are insensitive to exchange of perdeuterated solvent. Nonetheless, for such complexes, in which the nucleotide phosphate groups are equatorially

ANGLE-SELECTED ENDOR

105

coordinated to the presence of an axially coordinated solvent molecule could be demonstrated by ENDOR. Similarly, Schweiger and coworkers were able to demonstrate on the basis of ESEEM that benzaldehyde was axially coordinated through its carbonyl oxygen to in bis(1R-3- heptafluorobutyrylcamphorate)oxovanadium(IV), a Diels-Alder catalyst (Togni et al., 1993). By applying to the –7/2 parallel EPR absorption feature in pulsed ESEEM studies, they were able to show that the hfc components of an axial aldehyde group were very similar to those of axially coordinated to as illustrated in Fig. 5.

Figure 5. Stereo diagram of the coordination structure of in methanol determined on the basis of ENDOR and molecular modeling. The upper diagram illustrates the complex in stick skeletal form. Broken lines connect the inner-sphere methanol molecules coordinated to the vanadium. The lower diagram illustrates the complex in space-filling form (in the same projection as in the upper diagram), and was drawn to scale for van der Waals radii of 1.53 Å (C), 1.4 Å (O), 1.2 Å (H), and 1.35 Å (V). From Mustafi and Makinen (1988) with permission of the American Chemical Society.

The ion occupies a position between and according to ionic charge density defined on the basis of Z/R, where Z is the number of electrons and R the ionic radius (Williams, 1985). This characteristic undoubtedly accounts for its successful application as a paramagnetic probe

of

and

sites in proteins and nucleotides although

has been used as a substitute also for

and

sites in proteins

(Chasteen, 1981,

1983,1990). We have found

to

be a particularly

useful probe of

sites in proteins, revealing the chemical origins

of both equatorial and axial ligands (Mustafi and Nakagawa, 1994, 1996; Mustafi et al., 2000). In these studies the resonance features of in solution have provided an important basis for analyzing coordination environment in proteins. We have shown that the X-ray structure of

 

generally considered a model compound of

 

residues in proteins (Zell et al., 1985), does not account

for the

properties of the A and B isoforms of mammalian

106

DEVKUMAR MUSTAFI AND MARVIN W. MAKINEN

nephrocalcin. This protein containing four distinct

sites is the

important factor secreted into renal tubules retarding stone formation in the mammalian kidney. Isoforms A and B, exhibiting the tightest

affinity

contain 3-4 equivalents of Gla residues

each while

isoforms C and D have none and are associated with lower

 

affinity by two orders of magnitude (Nakagawa et al., 1981, 1983, 1985).

Fig.

6 compares the proton ENDOR spectra of

of

nephrocalcin isoforms B and D in natural and perdeuterated aqueous buffer. In each panel, the ENDOR spectrum of is also compared with that of the nephrocalcin complex. The results show that in isoform B, as in isoform A (Mustafi et al., 2000), only protein residues with nonexchangeable hydrogens are detected as ligands with complete exclusion of solvent water from the inner coordination sphere of the metal ion. The coordination geometry suggested by the complex places the Gla residues as bidentate ligands in the equatorial plane with an

axial

water (Zell et al., 1985). Clearly, the structure of the

 

complex cannot account for the coordination environment of

the

sites in nephrocalcin isoforms A and B. For isoforms C and D, on

the other hand, two water molecules are detected by ENDOR in the inner coordination sphere of the metal ion (Mustafi et al., 2000), but these isoforms do not contain Gla residues.

Nephrocalcin isoforms A and B undergo a more prominent conformational change upon binding or than do isoforms C and D, as measured through circular dichroism (Mustafi and Nakagawa, 1994, 1996; Mustafi et al., 2000). Under the assumption that the interior regions of isoforms A and B acquire a lower dielectric constant through the conformational change, becoming less polar, it is unusual that water is

excluded from the inner coordination sphere of the bound

ions. It is of

interest that parvalbumin contains two

sites, of which the high

affinity site

similarly has no inner-sphere coordinated water

(Kretsinger

and Nockolds, 1973).

Since nephrocalcin

hinders the

aggregation and growth of microcrystals of calcium oxalate into renal stones, elucidating the molecular basis by which stone formation is inhibited presents a challenging problem.

Since

binds only to the phosphate groups of nucleotides (Happe and

Morales,

1966) and is generally required for enzyme-catalyzed phosphoryl

transfer reactions in cells, it is important to determine structures of

 

complexes or to employ spectroscopic probes that closely

simulate

interactions with phosphate groups. In our laboratory we have

observed through EPR and ENDOR spectroscopy that

is coordinated

only to the phosphate groups of nucleotides, similar to

that observed for

 

complexes (Mustafi et al., 1992; Jiang and Makinen, 1995).

ANGLE-SELECTED ENDOR

107

Since it was possible to demonstrate in these studies that binding was inhibited by with no evidence of non-specific binding of the ion to the ribose hydroxyl groups or to the base hetero-atoms, it is likely that may serve as the most suitable paramagnetic probe to simulate interactions in spectroscopic studies.

Figure 6. Proton ENDOR spectra of complexes in frozen aqueous solutions. In the lefthand panel, spectra correspond to the following: a, : nephrocalcin isoform B (4:1 molar ratio) in protiated aqueous buffer; b, : isoform B (4:1 molar ratio) in deuterated aqueous buffer; and c, complex. In the right-hand panel, the spectra correspond to the same conditions but for nephrocalcin isoform D. In both sets of ENDOR spectra, the magnetic field was set to –3/2 perpendicular EPR absorption feature, and proton ENDOR absorptions from inner-sphere coordinated water molecules in axial and equatorial positions, labeled and respectively, are identified by stick diagrams. For the complex of isoform B, no resonance features for axial or equatorial coordinated water molecules are detected, as indicated by the broken vertical lines. For the of isoform D, proton ENDOR features for inner shell equatorial water molecules are seen, as indicated by the solid vertical lines between spectra a and b. In each panel ENDOR line pairs deriving from amino acid residues of the protein, labeled A-F, are indicated by stick diagrams with solid vertical lines for spectra a. The ENDOR line pairs are equally spaced about the free proton Larmor frequency of 13.9 MHz. The abscissa indicates the ENDOR shift. Reprinted from Mustafi et al. (2000) with permission of the CMB Association.

Of the five types of nucleic acid bases in DNA and RNA, guanine is unique because its nucleosides and nucleotides are capable of forming selfstructured assemblies in solution through hydrogen-bonding to yield G – G base pairs and G-quartets. The latter are square planar arrays of the guanine bases with Hoogsteen hydrogen bonding interactions with each other. These assemblies are important because the 3’-overhang regions of DNA strands in the cell are rich in guanine and serve as the point of attachment of the DNA strand to protein subunits comprising the mitotic spindle apparatus in cell

108 DEVKUMAR MUSTAFI AND MARVIN W. MAKINEN

division (Blackburn, 2000). It is thought that G-quartet formation in these regions of DNA strands may be important in binding to the protein.

It has long been known that G-quartet assemblies can be formed from 5’- GMP in solution as a square-planar array of hydrogen-bonded guanine bases (Gellert et al., 1962). Equilibria controlling the formation of G-quartets and of stacked quartets to form octets and higher order assemblies are sensitive to pH and the presence of sodium and potassium ions (Pinavaia et al., 1978). Although helical fibers of stacked quartets of 5'-GMP were characterized crystallographically (Zimmerman, 1976) and by infrared spectroscopy (Audet et al., 1991), the pucker of the ribose ring and conformation of the base moiety in these helical arrays were not established.

Jiang and Makinen (1995) demonstrated on the basis of NMR studies that the complex in the presence of excess 5’-GMP enters into self-structured quartet and octet assemblies through hydrogen-bonding like the free nucleotide. They were able to show that the ENDOR shifts of the protons assigned to the ribose moiety and to the 8-H position in the nucleic acid base were identical for the monomeric form of as well as for the metal-nucleotide complex incorporated into quartet and octet assemblies. This observation indicated that the conformation of the metal-nucleotide complex was unchanged upon its incorporation into G-quartet assemblies.

Table 3 summarizes the ENDOR transition frequencies and their assignments for the complex in solution. Fig. 7 illustrates the results of torsion angle search calculations. Only a small family of conformations accommodate the ENDOR-determined vanadiumproton distances within van der Waals hard-sphere constraints. Different conformations of the ribose ring were tested. The ENDOR determined electron-proton distances restricted the ribose conformation to a C3'- endo pucker. Modeling studies using the ENDOR distances as constraints were able to rule out the C2- endo conformation, which is the other prevalent conformation found for monomeric ribonucleotides (Saenger, 1984), On the other hand, for G-quartets formed with in solution, the guanine base was restricted to the anti conformation. The X-ray structure of double-stranded which forms hydrogen-bonded arrays of G- quartets in crystals, shows the guanine base to occupy both syn and anti conformations within each planar quartet array (Kang et al., 1992). However, the quality of the electron density map did not allow an unambiguous assignment of ribose pucker. In deoxyribonucleotides the absence of the 2’-hydroxyl group allows more conformational flexibility with respect to ring pucker than in ribonucleotides. The structure of the

complex incorporated into a G-quartet, as derived

ANGLE-SELECTED ENDOR

109

through EPR, ENDOR, and NMR investigations (Jiang and Makinen, 1995) is illustrated in Fig. 8.

Figure 7. Angle maps showing conformational space accessible to the 5’-GMP moiety in under van der Waals hard-sphere constraints only (low density dots) and upon application of the distance constraints in Table 3 (high density dots connected by broken lines to the conformer accommodating vanadium-proton distance constraints within their line width based uncertainties). Because of the closed ring structures of the nucleic acid base and of the ribose, only four dihedral angles define the conformation of 5’-GMP (standard designations shown in the left-hand structure). The right-hand structure depicts the ENDOR assigned conformation as C3’- endo. In the upper angle map, the axes correspond to 0–360°

of rotation for the dihedral angles

and

over which the search calculations were carried out in 1°

increments. The lower angle map shares axes for dihedral angles

and while the third axis

represents 0–360° of rotation for the dihedral angle