Daniel S. C. Yang

Bone recognition mechanism of porcine osteocalcin from crystal structure.

Osteocalcin is the most abundant noncollagenous protein in bone, and its concentration in serum is closely linked to bone metabolism and serves as a biological marker for the clinical assessment of bone disease. Although its precise mechanism of action is unclear, osteocalcin influences bone mineralization, in part through its ability to bind with high affinity to the mineral component of bone, hydroxyapatite. In addition to binding to hydroxyapatite, osteocalcin functions in cell signalling and the recruitment of osteoclasts and osteoblasts, which have active roles in bone resorption and deposition, respectively. Here we present the X-ray crystal structure of porcine osteocalcin at 2.0 Å resolution, which reveals a negatively charged protein surface that coordinates five calcium ions in a spatial orientation that is complementary to calcium ions in a hydroxyapatite crystal lattice. On the basis of our findings, we propose a model of osteocalcin binding to hydroxyapatite and draw parallels with other proteins that engage crystal lattices.

Antifreeze Proteins in Winter Rye Are Similar to Pathogenesis-Related Proteins

The ability to control extracellular ice formation during freezing is critical to the survival of freezing-tolerant plants. Antifreeze proteins, which are proteins that have the ability to retard ice crystal growth, were recently identified as the most abundant apoplastic proteins in cold-acclimated winter rye (Secale cereale L.) leaves. In the experiments reported here, amino-terminal sequence comparisons, immuno-cross-reactions, and enzyme activity assays all indicated that these antifreeze proteins are similar to members of three classes of pathogenesis-related proteins, namely, endochitinases, endo-[beta]-1,3-glucanases, and thaumatin-like proteins. Apoplastic endochitinases and endo-[beta]-1,3-glucanases that were induced by pathogens in freezing-sensitive tobacco did not exhibit antifreeze activity. Our findings suggest that subtle structural differences may have evolved in the pathogenesis-related proteins that accumulate at cold temperatures in winter rye to confer upon these proteins the ability to bind to ice.

Extraction and Isolation of Antifreeze Proteins from Winter Rye (Secale cereale L.) Leaves

Apoplastic extracts of cold-acclimated winter rye (Secale cereale L. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaCl2 or MgSO4. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. The five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11- and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. The amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Glu/Gln, Ser, Thr, Gly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. These polypeptides were examined with antisera to other cystine-containing antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and insects.

Identification of the Ice-Binding Surface on a Type III Antifreeze Protein with a “Flatness Function” Algorithm

Antifreeze proteins (AFPs) adsorb to surfaces of growing ice crystals, thereby arresting their growth. The prevailing hypothesis explains the nature of adsorption in terms of a match between the hydrophilic side chains on the AFPs ice-binding surface (IBS) and the water molecules on the ice surface. The number and spatial arrangement of hydrogen bonds thus formed have been proposed to account, respectively, for the binding affinity and specificity. The crystal structure of a type III AFP from ocean pout (isoform HPLC-3) has been determined to 2.0-A resolution. The structure reveals an internal dyad motif formed by two 19-residue, loop-shaped elements. Based on of the flatness observed on the type I alpha-helical AFPs IBS, an automated algorithm was developed to analyze the surface planarity of the globular type III AFP and was used to identify the IBS on this protein. The surface with the highest flatness score is formed by one loop of the dyad motif and is identical to the IBS deduced from earlier mutagenesis studies. Interestingly, 67% of this surface contains nonpolar solvent-accessible surface area. The success of our approach to identifying the IBS on an AFP, without considering the presence of polar side chains, indicates that flatness is the first approximation of an IBS. We further propose that the specificity of interactions between an IBS and a particular ice-crystallographic plane arises from surface complementarity.

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing.

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.

Fatty-acid chain tilt angles and directions in dipalmitoyl phosphatidylcholine bilayers

X-ray diffraction has been applied to determine the various tilt angles and directions (if any) which can be assumed by oriented gel phase multilayers of dipalmitoyl phosphatidylcholine (DPPC) as a function of hydration. We report for the first time that oriented DPPC multilayers with a repeat spacing (d-spacing) of 55.2A at 25 degrees C and 0% relative humidity (RH) have hydrocarbon chains tilted at an angle theta of 21.5 degrees with respect to the bilayer normal. In addition, the chains are tilted along one of the bisectors (omega = 0 degrees) of the hexagonal lattice (8 wide-angle maxima, 2 unique), a phase not previously reported in DPPC studies. At 100% RH, the chain tilt angle and d-spacing increased to approximately 29.0 degrees and 58.9A, respectively. Since at 100% RH only 4 wide-angle maxima are observed, we analyze the data on the assumption that the hydrocarbon chains may rotate independently of the hexagonal lattice (omega = 0-30 degrees), at a fixed chain tilt angle theta (Stamatoff, J.B., et al. 1979. Biophys. J. 25:253-262). The largest observed angle phi made by the wide-angle maxima with the equator is 29.5 degrees corresponding to a theta of approximately 32.6 degrees (omega avg. = 24 degrees) and the sample having a d-spacing of 64.0 A (excess water condition). Finally, theta remains relatively constant (approximately 21.5 degrees) up to a RH of approximately 45% and a d-spacing of 57.8A, after which, with increases in RH, theta increases to a maximum of 32.6 degrees.

Ca2+-dependent antifreeze proteins. Modulation of conformation and activity by divalent metal ions.

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. The type II fish AFPs of Atlantic herring and smelt are unique among known AFPs in their requirement of a cofactor for antifreeze activity. These AFPs are homologous with the carbohydrate-recognition domains of Ca2+-dependent (C-type) lectins and require Ca2+ for their activity. To investigate the role of metal ions in the structure and function of type II AFPs, the binding of Ca2+ and other divalent cations to herring AFP was investigated. Binding studies using 45Ca2+ demonstrated that the AFP has a single Ca2+-binding site with a Kd of 9 μM. Proteolysis protection studies and measurement of antifreeze activity revealed a conformational change from a protease-sensitive and inactive apoAFP to a protease-resistant active AFP upon Ca2+ binding. Other divalent metal ions including Mn2+, Ba2+, and Zn2+ bind at the Ca2+-binding site and induce a similar change. A saturatable increase in tryptophan emission intensity at 340 nm also occurred upon Ca2+ addition. Whereas antifreeze activity appeared normal when Ca2+ or Mn2+ were bound, it was much lower in the presence of other metal ions. When Ba2+ was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca2+ and, consequently, adopts a conformation that is essential for its ice-binding activity.

Structure and Absolute Configuration of (R)-Baclofen Monohydrochloride

C~oHI3C1NO+.C1 -, M, = 250.13, ortho- rhombic, P2~2t2 ~, a = 6.373 (1), b = 7.318 (2), c = 25.699 (5) A, 2(Cu) = 1.54180 A, V= 1198.5 A 3, Z = 4, D c- 1.386 g cm -3, ~ = 47.35 cm-k The phase problem was solved by the direct method (MULTAN 78); R(F) -- 0.029 for 1169 reflections. The molecules are linked into infinite chains along the b axis by hydrogen bonding. There is no significant ring stacking. Introduction. Baclofen (y-amino-fl-(p-chlorophenyl)- butyric acid) is a derivative of the inhibitory neuro- transmitter y-aminobutyric acid (GABA) but, unlike GABA, it can cross the blood/brain barrier (Birkmayer, 1972). It has been shown that baclofen reduces excitatory transmitter effects, especially sub- stance P (Pier & Zimmerman, 1973; Polc & Haefely, 1976; Potashner, 1979; Saito, Konishi & Otsuka, 1975). Baclofen has become the drug of choice in the treatment of spasticity of spinal origin due to its antispastic efficacy at doses which do not produce Cl sedation, its low frequency of serious side effects and its 0(1) lack of organ toxicity (Sachais & Logue, 1977). Recent o(2) studies have shown baclofen to be a promising new N drug in the treatment of the paroxysmal pain of c(1) trigeminal neuralgia (Fromm, Terrence, Chattha & c(2) Glass, 1980). c(4) White, tabular crystals were grown from water by c(5) slow evaporation. The space group was uniquely C(6) determined from Weissenberg photographs as P212~21. C(7) The unit-cell parameters were obtained from a least- c(8) squares fitting of the setting angles for 12 reflections c(10) measured on a Picker FACS-1 diffractometer. The H(O) intensity data were collected using graphite-mono- H 1(2) chromated Cu Ka radiation with the 0:20 scan H2(2) technique. Within the 20 range of 5.0 ° to 125.0 °, 68 Hl(4) out of 1 169 reflections were considered unobserved by H2(4) the criterion I < 3o(1). The E map generated by H(6) MULTAN 78 (Main, 1978) revealed positions of two H(7) CI atoms. All C and N atoms were located from a H(10) subsequent Fourier synthesis. The structure was refined by the full-matrix least-squares refinement procedures and all H atoms (with the exception of those of the NH 3 group) were located in a difference Fourier map. A close look at the difference Fourier map around N showed vague positions of three H atoms. The coordinates of these H atoms were fixed from consideration of the hydrogen bonding to CI-. In the final stages of the refinement all atoms except H were refined with anisotropic temperature factors. The final R factor is 0.029 while the weighted R factor,

Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112–160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.

STUDIES OF A PUTATIVE ICE-BINDING MOTIF IN WINTER FLOUNDER SKIN-TYPE ANTI-FREEZE POLYPEPTIDE

Winter flounder contains two distinct anti‐freeze protein isoforms, which are the liver‐type extracellular anti‐freeze proteins and the skin‐type intracellular anti‐freeze protein. The skin‐type anti‐freeze proteins exhibit lower anti‐freeze activities than the liver‐type isoforms and this might be due to their lacking complete ice‐binding motifs. One of the skin‐type anti‐freeze proteins, skin‐type anti‐freeze protein‐3, does contain putative overlapping ice‐binding motifs with the sequences ‘‐K—DT‐’ and ‘‐DT–K‐’. Synthetic anti‐freezes containing 0–3 repeats of the ‘‐DT–K‐’ motif were tested for stability and activity. Loss of the single ‘‐DT–K‐’ of skin‐type anti‐freeze protein‐3 increases the anti‐freeze activity and increasing the number of motifs to two or three lowers the activity. The decrease in activity with an increasing frequency of the motif correlates with a decrease in the helical content of these peptides at 0°C.