Hemoglobin allostery: resonance Raman spectroscopy of kinetic intermediates.

The end states, R and T, of the allosteric transition in hemoglobin (Hb) are structurally well characterized, but there is little information on intermediate structures along the allosteric pathway. These intermediates were examined by means of time-resolved resonance Raman spectroscopy in the nanosecond-to-microsecond interval after HbCO photolysis. Complementary spectra of the heme group and of the tyrosine and tryptophan residues were recorded during laser excitation at 436 and 230 nanometers. These spectra reveal a sequence of interleaved tertiary and quaternary motions during the photocycle, motions involving the proximal and distal helices, and the alpha 1 beta 2 subunit interface. This sequence leads to a modified form of the T state, in which the alpha 1 beta 2 interface is deformed as a result of two carbon monoxide molecules binding to the same dimer within the tetramer.

Where plants make oxygen: a structural model for the photosynthetic oxygen-evolving manganese cluster.

In the photosynthetic evolution of oxygen, water oxidation occurs at a catalytic site that includes four manganese atoms together with the essential cofactors, the calcium and chlorine ions. A structural model and a determination of the manganese oxidation states based on x-ray absorption spectroscopy are presented. The salient features, in both higher plants and cyanobacteria, are a pair of di-mu-oxo bridged manganese binuclear clusters linked by a mono-mu-oxo bridge, one proximal calcium atom, and one halide. In dark-adapted samples, manganese occurs in oxidation states (III) and (IV). Data from oriented membranes display distinct dichroism, precluding highly symmetrical structures for the manganese complex.

Modeling the hemoglobin switchpoint with cyanomet valency hybrids: Raman spectroscopic probes of tertiary and quaternary structure.

Hybrid hemoglobins with cyanomet hemes in the alpha or in the beta chains have been investigated by resonance Raman (RR) spectroscopy, using ultraviolet (230 nm) and visible (441.6 nm) excitation. For the CO adducts, the UVRR spectra are identical with that of native HbCO, showing the tyrosine and tryptophan signals to be insensitive to ligand substitution within the R state. In the absence of CO, the doubly ligated hybrids show differences in the UVRR spectra, relative to the CO adducts, which are the superposition of two difference spectra: (1) the T-R difference spectrum obtained by subtracting the spectrum of HbCO from that of deoxyHb and (2) a perturbed R state spectrum, characteristic of deligated chains within the R state. These spectra arise from alterations, respectively, in the quaternary contacts of interface aromatic residues and in the tertiary contacts of interior aromatic residues. From the amplitudes of the difference spectra, the T state population was determined to be 30% for (alpha FeII beta FeIIICN)2 and 43% for (alpha FeIIICN beta FeII)2, in good agreement with the kinetic analysis of Cassoly and Gibson [Cassoly, R., & Gibson, Q. H. (1972) J. Biol. Chem. 247, 7332]. Addition of inositol hexaphosphate (IHP) increased the T state population, but only by a modest amount, to 40 and 53%, respectively, in contrast to the frequent assumption that the T state conversion is quantitative in the presence of IHP. Since current understanding of the quaternary state dependence of the Fe-histidine stretching frequency is based on that assumption, the RR band envelope for this vibration was reexamined.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein.

SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.

Solubility of fluoromethemoglobin S: effect of phosphate and temperature on polymerization.

The polymerization properties of the fully liganded fluoromet derivative of hemoglobin S (FmetHb S) were investigated by electron microscopy and absorption spectroscopy. Polymerization progress curves, as measured by increasing sample turbidity at 700 nm, exhibit a delay time (t(d)) consistent with the double nucleation mechanism. The pattern of fiber growth, as monitored by electron microscopy, is also indicative of a heterogeneous nucleation process, and dimensions of the fibers were found to be comparable to that of deoxyHb S. The polymerization rate constant (1/t(d)) depends exponentially on Hb S concentration, and the size of the homogeneous and heterogeneous nuclei also depend on FmetHb S concentration. As for deoxyHb S, higher concentrations of protein and phosphate favor fiber formation, while lower temperatures inhibit polymerization. Solubility experiments reveal, however, that eight times more FmetHb S is required for polymerization. The current studies further show that reaction order is independent of phosphate concentration if Hb S activity and not concentration is considered. The allosteric effector, inositol hexaphosphate (IHP), promotes fiber formation, and temperature-dependent reaggregation of FmetHb S suggests that IHP stabilizes pregelation aggregates. These studies show that FmetHb S resembles deoxyHb S in many of its polymerization properties; however, IHP-bound FmetHb S potentially provides a unique avenue for future studies of the early stages of Hb S polymerization and the effect of tertiary and quaternary protein structure on the polymerization process.

Nucleic Acid Structure Investigated by UV Resonance Raman Spectroscopy: Protonation Effects and A-Tract Structure.

Abstract UV resonance Raman (UVRR) spectroscopy has been used to investigate the three purine bases, adenine, guanine and inosine, as a function of pH. Excitation wavelengths of 260 and 210 nm were used to probe the in-plane ring stretching frequencies and the exocyclic functional groups, respectively. These studies are suggestive that tautomeric forms can be stabilized at low and high pH values and these forms can be identified using UVRR spectroscopy. At pH values ≤5.0, a band at 1693 cm (-1) is observed in the UVRR spectra of dAMP, which is suggestive of the imino protonated tautomer. At pH values of 10.0 and above both dGMP and IMP show evidence for forming the enolate tautomer, by the loss in intensity of the C=O stretching mode at 1686 cm(-1). The protonated forms of dGMP and dAMP exhibit distinct Raman bands at approximately 1460 and 1561 cm(-1) and we suggest that these protonated states can be identified using UVRR spectroscopy. Most distinctively, the -NH(2) scissors mode of dGMP and dAMP shifts up in frequency and increases in intensity as the pH is decreased. Interestingly, these features are also observed in a comparison of an A-tract containing dodecamer with a non A-tract dodecamer. In particular, a frequency upshift of the -NH(2) scissors mode and a mode at 1466 cm(-1) is observed. Because of the resonance enhancement and the similarities to the protonated dAMP spectrum, these features are attributed to the dA residues in the A-tract. It is suggested that these spectral features may be characteristic of 'bent' DNA.

R and T states of fluoromethemoglobin probed by ultraviolet resonance Raman spectroscopy.

Ultraviolet resonance Raman spectra are reported for the fluoride complex of methemoglobin (metHbF), with and without added inositol hexaphosphate (IHP), an allosteric effector known to stabilize the T quaternary structure. Environmental changes in Trp and Tyr residues give rise to the difference features, which are similar to those observed in the difference spectrum between deoxyHb and the CO adduct, consistent with T-state formation in metHbF in the presence of IHP. There are, however, important differences. The intensities of the difference signals are attenuated by about one-third, indicating a lower T-state population in the IHP-bound metHbF than in native deoxyHb. And a new signal is seen which arises from the interior tryptophans, probably reflecting a change in their H-bond status associated with the presence of fluoride as the sixth heme ligand in the T state. Implications of these results for the nature of the molecular forces opposing ligation in the T state are discussed.

Nucleotide binding activity of SecA homodimer is conformationally regulated by temperature and altered by prlD and azi mutations.

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4 degrees C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi- and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi- and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.

New light on allostery: dynamic resonance Raman spectroscopy of hemoglobin kempsey.

On the basis of static and time-resolved resonance Raman spectroscopy of HbA and of a mutant, HbK (Dalpha99N), a specific reaction coordinate is proposed for the allosteric transition in human hemoglobin. The heme is held between proximal (F) and distal (E) helices, whose orientation is responsive to forces generated by ligation and deligation. The E and F helices are in turn tethered via H-bonds to the A and H helices. These outer helices follow the E-F motion, thereby repositioning the N- and C-termini, which form the intersubunit salt bridges in the T quaternary structure. When the T state interface is weakened by Asp --> Asn substitution at a quaternary H-bond (HbK), the Fe-His bond is relaxed and becomes responsive to allosteric effectors. The same E-F motion is observed in HbK, but the A-H following motion is delayed, relative to HbA, as is the Asn H-bond formation.

A UV resonance Raman study of d(A(+)-G)10, a single-stranded helix without stacked or paired bases.

UV resonance Raman spectroscopy has been utilized to directly observe structural features of the recently described nucleic acid single-stranded helix d(A(+)-G)10. An absence of base stacking is confirmed by invariant hypochromic ratios of dominant vibrational modes for the oligomer relative to its constituent monomers as the structure is thermally denatured. The N1 of dA residues is protonated, as determined by similarity to the ring-stretching vibrations for protonated adenine and its derivatives. Selective resonance enhancement of Raman vibrational modes from dA and dG residues shows frequency shifts upon thermal denaturation that confirm the participation of the exocyclic amino of dA but not dG residues in H-bonding. Conformationally sensitive glycosyl bond modes suggest anti residue conformations.

HU binding to DNA: evidence for multiple complex formation and DNA bending.

HU, a nonspecific histone-like DNA binding protein, participates in a number of genomic events as an accessory protein and forms multiple complexes with DNA. The HU-DNA binding interaction was characterized by fluorescence, generated with the guanosine analogue 3-methyl-8-(2-deoxy-beta-D-ribofuranosyl)isoxanthopterin (3-MI) directly incorporated into DNA duplexes. The stoichiometry and equilibrium binding constants of complexes formed between HU and 13 and 34 bp DNA duplexes were determined using fluorescence anisotropy and analytical ultracentrifugation. These measurements reveal that three HU molecules bind to the 34 bp duplexes, while two HU molecules bind to the 13 bp duplex. The data are well described by an independent binding site model, and the association constants for the first binding event for both duplexes are similar (approximately 1 x 10(6) M(-1)), indicating that HU binding affinity is independent of duplex length. Further analysis of the binding curves in terms of a nonspecific binding model is indicative that HU binding to DNA exhibits little to no cooperativity. The fluorescence intensity also increases upon HU binding, consistent with decreased base stacking and increased solvent exposure of the 3-MI fluorescence probe. These results are suggestive of a local bending or unwinding of the DNA. On the basis of these results we propose a model in which bending of DNA accompanies HU binding. Up to five complex bands are observed in gel mobility shift assays of HU binding to the 34 bp duplexes. We suggest that protein-induced bending of the DNA leads to the observation of complexes in the gel, which have the same molecular weight but different relative mobilities.

Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA.

Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs.

A UV resonance Raman study of hairpin dimer helices of d(A-G)10 at neutral pH containing intercalated dA residues and alternating dG tetrads.

The structure of the oligonucleotide d(A-G)10 in 0.6 M Na+, pH 7.0 has been investigated with UV resonance Raman (UVRR) spectroscopy. Variable wavelength excitation was used to distinguish the spectral contributions of dG and dA residues. Both classes of residues show UVRR hyperchromism with increasing temperature, reflecting unstacking of the bases. The dG residues melt relatively cooperatively with a Tm of approximately 42 degrees C. Unstacking is non-cooperative for the dA residues, increasing linearly between 4 and 80 degrees C. G-tetrads at low temperature are indicated by UVRR frequency shifts of modes associated with C6=O and C2-NH2 of the dG residues, and of vibrations involving N7, all sites of H-bonding. However, there are no indications of interbase H-bonds for the dA residues, showing they do not form H-bonded tetrads. Most of the bases are oriented anti about the glycosyl bond, but at 4 degrees C a fraction of the residues are syn. These results, together with the findings by Shiber et al. [Shiber,M.C., Braswell,E.H., Klump,H. and Fresco,J.R. (1996) Nucleic Acids Res. 24, 5004-5012] that d(A-G)10 under comparable conditions has the molecular weight of a dimer, support a model in which two hairpins interact to form a helical structure with G-tetrads and intercalated dA residues.

Evidence for the proximity of calcium to the manganese cluster of photosystem II: determination by X-ray absorption spectroscopy.

The photosynthetic oxygen-evolving complex contains a cluster of four manganese atoms and requires both Ca and Cl for activity. The question of Ca proximity to the Mn cluster has been investigated by performing Mn X-ray absorption experiments on native samples of photosystem II (PS II) and on samples depleted of Ca and reconstituted by either Ca or Sr. Analysis of X-ray K-edge spectra demonstrates no significant differences in oxidation state or symmetry between Ca- and Sr-reactivated preparations. Differences are observed in the extended X-ray absorption fine structure (EXAFS). The amplitude of a Fourier transform peak due to scatters at distances greater than 3 A is larger for samples reactivated with strontium than for calcium-reactivated samples. Taking into account the stoichiometry of Mn and Ca atoms in PS II, and considering physically reasonable structures, curve-fitting analyses of the EXAFS data using FEFF5-calculated parameters favor a model where both manganese and calcium (or strontium) scatterers contribute to the Fourier peak at approximately 3 A. Other models for the approximately 3 A peak with multiple Mn-Mn interactions or multiple Mn-Ca(Sr) interactions can also be fit to the data, but are considered less likely. This result provides confirmation for the structural proximity of Ca to the Mn cluster suggested previously [Yachandra, V. K., et al. (1993) Science 260, 675-679]. Possible structural arrangements for a calcium-binding site are discussed.

Orientation of the oxygen-evolving manganese complex in a photosystem II membrane preparation: an X-ray absorption spectroscopy study.

X-ray absorption spectroscopy has been performed on oriented photosystem II membrane particles isolated from spinach. Structural features of the tetranuclear Mn cluster and the orientation of the cluster with respect to the lipid bilayer were determined in both the S1 and S2 states of the Kok cycle. Variation of the sample orientation with respect to the X-ray e-vector yields highly dichroic K-edge and extended X-ray absorption fine structure spectra (EXAFS), indicative of an asymmetric tetranuclear cluster. Mn-Mn vectors at 2.72 and 3.38 A can be resolved from these measurements using quantitative analysis. The 2.72-A vector, consisting of at least two component vectors, is oriented at an average angle of 60 degrees +/- 7 degrees to the membrane normal, with an average of 1.1 +/- 0.1 interactions per Mn atom. The 3.38-A vector, most probably an average of two vectors, makes an angle of 43 degrees +/- 10 degrees with respect to the membrane normal, with an average of 0.45 +/- 0.07 backscatterer per Mn atom. Upon advance to the S2 state, the orientation of these vectors and the average numbers of backscatterers are approximately invariant. Analysis of more subtle features of the EXAFS reveals changes accompanying this S-state advance that are consistent with the oxidation of Mn during this transition. However, the dominant structural features of the oxygen-evolving complex remain constant in the S1 and S2 states. The structure of the Mn complex and the orientation of the complex in the membrane within the context of dichroism of the X-ray absorption data are discussed.