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Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.

The physiological function of the prion protein (PrP) remains enigmatic despite its established involvement in the pathogenesis of spongiform encephalopathies. PrP is a glycolipid-anchored membrane protein, which constitutively recycles between the cell surface and an endosomal compartment. The N-terminal region of PrP contains a four tandem repeat (OP4) of the octapeptide PHGGGWGQ (OP) that binds and reduces Cu(II) ions. We have examined the kinetic properties of the OP4-mediated Cu(II) reduction and found that OP4 exhibits the highest reduction activity around pH 6.5, close to the pH in early endosomes. All four OP units and at least one tryptophan side chain are essential for Cu(II) reduction. The reaction is described by an uncompetitive substrate inhibition mechanism involving a 1:1 Cu(II)-OP4 active intermediate. Structural analysis by Raman spectroscopy has revealed that the Cu(II) ion is coordinated by four histidine Ntau atoms in the active intermediate and the feasibility of formation of this intermediate correlates with the Cu(II) reduction over a pH range from 5.0 to 8.2. Molecular mechanics calculations suggest that two tryptophan residues of OP4 are located near the Cu(II) site, being consistent with the importance of redox-active tryptophan in the Cu(II) reduction. PrP has been proposed to capture Cu(II) ions in the extracellular space and release them in the endosome. The results of this study strongly suggest that PrP also plays a role in the reduction of captured Cu(II) ions prior to their transfer to Cu(I)-specific intracellular copper trafficking proteins.

Molecular dynamics analyses were performed to examine conformational changes in the C-domain of calmodulin and the N-domain of troponin C induced by binding of Ca(2+) ions. Analyses of conformational changes in calmodulin and troponin C indicated that the shortening of the distance between Ca(2+) ions and Ca(2+) binding sites of helices caused widening of the distance between Ca(2+) binding sites of helices on opposite sides, while the hydrophobic side chains in the center of helices hardly moved due to their steric hindrance. This conformational change acts as the clothespin mechanism.

Ultraviolet resonance Raman spectra of ribosyl C(1')-deuterated guanosine and adenosine were measured. Most in-plane vibrations of the purine rings in the 1420-1100-cm -1 region showed frequency upshifts upon C(1')-deuteration, while those in the 1100-700-cm -1 region showed downshifts. The purine ring vibrations above 1450 cm -1 were unaffected. The frequency shifts associated with the C(1')-deuteration are explained by assuming couplings of the purine vibrations with a ribose C(1')-H bending mode, which involves a hydrogen motion in the plane of N(9)-C(1')-H

Ultraviolet resonance Raman spectra of phytochrome in the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr) are reported. The spectra excited at 240-nm provide structural information about the protein part of phytochrome. The protein contains only a very small amount of β-sheet structure and most of the tyrosine side chains are located in hydrophobic environments. Indole rings of tryptophan (Trp) interact with neighboring groups in the Pr form and these interactions become weaker with the conversion from Pr to Pfr. Some Trp side chains of Pfr are surrounded by aliphatic groups but such is not the case in Pr. These changes in the environment occur at the same time as changes in orientation of Trp side chains. Our observations suggest that interactions between Trp residues and the tetrapyrrolic chromophore occur in the Pr form and that the strength of these interactions diminishes in the Pfr form.

Distamycin A (Dst) is an antibiotic which binds to the minor groove of double-stranded DNA at A/T-rich regions. We have examined the affinity and mode of Dst binding to DNA duplexes containing a conserved A/T core and variable terminal A/T regions by using circular dichroism spectroscopy. The observed circular dichroism spectra were analyzed by singular value decomposition and fitted to a two-step binding model. The result clearly shows a correlation between the affinity for Dst and the preference for Dst-DNA 1:1 binding over 2:1 binding. The A/T stretches that prefer 1:1 binding form high-affinity 1:1 complexes, whereas those preferring 2:1 binding form stable 2:1 complex with low overall affinities. The terminal A/T residues of the Dst binding region play an important role in the stabilization/destabilization of the 1:1 and 2:1 complexes, resulting in a terminal residue-dependent variation of the binding affinity and the binding mode preference.

The BM2 protein of influenza B virus forms a transmembrane proton channel essential for the virus infection. We investigated the structure and mechanism of the BM2 proton channel by using a 31-mer peptide (BM2-TMP) representing the putative transmembrane domain of BM2, with special focus on His19, Trp23 and His27. Like the full-length protein, BM2-TMP formed a transmembrane proton channel activated at acidic pH with a midpoint of transition at pH 6.4 +/- 0.1. Mutation of His19 to Ala almost abolished the channel activity, whereas the His27-to-Ala mutant retained partial activity. The proton selectivity of the channel was lost upon substitution of Phe for Trp23. Comparison of CD, fluorescence and Raman spectra measured for wild-type and mutated BM2-TMP at varied pH showed the pK(a) of the imidazole ring to be approximately 6.5 for His19 and approximately 7.6 for His27. Analysis of the pH-dependent fluorescence and Raman intensities suggested the occurrence of cation-pi interaction between the protonated imidazole ring of His and the indole ring of Trp. The His19-Trp23 cation-pi interaction below pH 6.5 is likely to trigger the opening of the proton channel, whereas His27 is not essential but enhances the channel activity through interaction with Trp23, which constitutes the proton-selective gate.

The cation-pi interaction, a noncovalent interaction of electrostatic nature between a cation and an electron-rich pi system, is increasingly recognized as an important force that influences the structures and functions of molecules including proteins. Unlike other metal cations, the transition metal cation Cu2+ is not regarded to take part in a cation-pi interaction because Cu2+ tends to oxidize the pi electron system, in particular that of Trp, and to introduce covalency in the metal-pi electron interaction. This paper reports the first spectral evidence for the cation-pi interaction between Cu2+ and Trp. The Cu2+ ion bound to the amino N-terminal Cu2+/Ni2+ binding motif composed of three amino acid residues interacts with the indole ring of the fourth Trp residue in a noncovalent manner. The Cu2+-Trp interaction produces a distinct negative band at 223 nm in circular dichroism (CD), which disappears upon mutation or depletion of the Trp residue or upon replacement of the Cu2+ ion by Ni2+. In UV absorption, a pair of negative/positive intensity changes is generated at 222/231 nm by the Cu2+-Trp interaction, being consistent with the previous observations on the indole ring interacting with K+ or a cationic His imidazole ring. The negative CD band around 223 nm is characteristic of the Cu2+-Trp pair and may be useful as a marker of the Cu2+-Trp cation-pi interaction. Coordination of negatively charged ligands to Cu2+ is suggested to be important for the cation to be involved in a cation-pi interaction.

The ClC chloride channels control the ionic composition of the cytoplasm and the volume of cells, and regulate electrical excitability. Recently, it has been proposed that prokaryotic ClC channels are H + –Cl − exchange transporter. Although X-ray and molecular dynamics (MD) studies of bacterial ClC channels have investigated the filter open–close and ion permeation mechanism of channels, details have remained unclear. We performed MD simulations of ClC channels involving H + , Na + , K + , or H 3 O + in the intracellular region to elucidate the open–close mechanism, and to clarify the role of H + ion an H + –Cl − exchange transporter. Our simulations revealed that H + and Na + caused channel opening and the passage of Cl − ions. Na + induced a bead-like string of Cl − –Na + –Cl − –Na + –Cl − ions to form and permeate through ClC channels to the intracellular side with the widening of the channel pathway.

Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability.

Cyclic AMP (cAMP) receptor protein (CRP) forms 1:1 and 1:2 complexes with cAMP, and the former complex is considered to be the most active form of CRP in binding to specific DNA sequences and in modulating gene transcription by RNA polymerases. We examine the cAMP binding modes and structural changes of CRP upon cAMP binding by UV resonance Raman spectroscopy. The Raman spectra of CRP-(cAMP)1 and CRP-(cAMP)2 extracted from those of CRP–cAMP mixtures at varied mixing ratios clearly show that the hydrogen bonding state and the conformation of cAMP in both complexes in solution are very similar to those found in the X-ray crystal structure of CRP-(cAMP)2, which is evidence that the cAMP binding mode does not differ between the two complexes. The environmental hydrophobicity of Trp85 monitored by UV resonance Raman intensity shows a significant decrease upon binding of the first cAMP molecule, whereas no further change occurs in the second cAMP binding step. The environmental change of Trp85 suggests an opening of the cleft between the N-terminal cAMP and C-terminal DNA binding domains in the process of CRP activation by binding of a single cAMP molecule. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 186–196, 2002

Interleukin 7 (IL-7) promotes both thymopoiesis and mature T lymphocyte survival and proliferation. In experimental murine models of hematopoietic stem cell transplantation (HSCT), reconstitution of the naïve T cell population from donor-derived HSC is enhanced by IL-7 treatment. Since HSC products for transplantation may also contain IL-7-responsive mature T lymphocytes, IL-7 treatment in allogeneic BMT could lead to exacerbation of graft-versus-host disease (GVHD). Using IL-7 deficient murine models, we have previously shown that IL-7 mediates survival of mature T lymphocytes and is necessary for pathogenesis of GVHD. In the present study, we determined whether GVHD could be prevented by blockade of IL-7 signaling with an anti-murine IL-7 receptor alpha chain (IL-7Rα ) antibody. In order to induce GVHD, C57/BL6 (H2Kb) recipient mice were lethally irradiated (1300 cGy) and co-transplanted with 4x106 lymph node (LN) and 1x106 T cell depleted (TCD) BM cells from MHC-mismatched allogeneic Balb/C (H2Kd) donor mice. Following transplantation, the allogeneic BMT recipients were intraperitonally injected with either the anti-murine IL-7Rα antibody, SB14 (100μg/mouse/week) or PBS for 5 weeks. GVHD-related mortality and morbidity were decreased in allogeneic recipients treated with anti-IL-7Rα anti-body compared to the PBS treated groups. Anti-IL-7Rα antibody treatment significantly increased survival in the B6 mice (70%, n=20) for up to 5 months when compared to the PBS treated B6 recipients (20%, n=20 [p