Biomedical subjects

G W Han

Publications and source records attributed to G W Han.

9 recordsLinked to original sources

PubMed1997

Structure of a DNA analog of the primer for HIV-1 RT second strand synthesis.

The non-self-complementary DNA decamer C-A-A-A-G-A-A-A-A-G/C-T-T-T-T-C-T-T-T-G is a DNA/DNA analogue of a portion of the polypurine tract or PPT, which is a RNA/DNA hybrid that serves as a primer for synthesis of the (+) DNA strand by HIV reverse transcriptase (RT), and which is not digested by the RNase H domain of reverse transcriptase following (-) strand synthesis. The same unusual conformation that eludes RNase H, thought to be a change in width of minor groove, may also be responsible for the inhibition of HIV RT by minor groove binding drugs such as distamycin and their bis-linked derivatives. The present X-ray crystal structure of this DNA decamer exhibits the usual properties of A-tract B-DNA under biologically relevant conditions: large propeller twist of base-pairs, narrowed minor groove, and a straight helix axis. Groove narrowing is fully developed in the A-A-A-A region, but not in the A-A-A region, which previous investigators have proposed as being too short to exhibit typical A-tract properties. The RNA/DNA hybrid produced by HIV reverse transcriptase during (-) strand synthesis presumably forms a "heteromerous" or H-helix with narrower minor groove than an A-helical RNA/RNA duplex. If the narrowing of minor groove in A-tract H-helices is comparable to that seen in A-tract B-helices, then the narrowed minor groove of the polypurine tract could make the second primer site both (1) impervious to RNase H digestion, and (2) susceptible to inhibition by minor groove binding drugs.

Crystallography, X-Ray↗

PubMed1996

Identifying the physiological electron transfer site of cytochrome c peroxidase by structure-based engineering.

A technique was developed to evaluate whether electron transfer (ET) complexes formed in solution by the cloned cytochrome c peroxidase [CcP(MI)] and cytochromes c from yeast (yCc) and horse (hCc) are structurally similar to those seen in the respective crystal structures. Site-directed mutagenesis was used to convert the sole Cys of the parent enzyme (Cys 128) to Ala, and a Cys residue was introduced at position 193 of CcP(MI), the point of closest contact between CcP(MI) and yCc in the crystal structure. Cys 193 was then modified with a bulky sulfhydryl reagent, 3-(N-maleimidylpropionyl)-biocytin (MPB), to prevent yCc from binding at the site seen in the crystal. The MPB modification has no effect on overall enzyme structure but causes 20-100-fold decreases in transient and steady-state ET reaction rates with yCc. The MPB modification causes only 2-3-fold decreases in ET reaction rates with hCc, however. This differential effect is predicted by modeling studies based on the crystal structures and indicates that solution phase ET complexes closely resemble the crystalline complexes. The low rate of catalysis of the MPB-enzyme was constant for yCc in buffers of 20-160 mM ionic strength. This indicates that the low affinity complex formed between CcP(MI) and yCc at low ionic strength is not reactive in ET.

Animals↗

PubMed1996

Abnormal desmoglein expression by squamous cell carcinoma cells.

Abnormal expression of cell adhesion molecules and related proteins has been observed in various carcinoma cells. We compared expression patterns of desmosomal cadherins, E-cadherin, and cytoplasmic plaque proteins of four different human squamous cell carcinoma cell lines and in vivo squamous cell carcinoma cells with those of normal human keratinocytes. Unlike normal human keratinocytes, the squamous cell carcinoma cells, both in culture and in vivo, exhibited diminished or unusual expression of desmoglein 3 and desmoglein 1, which bear pemphigus vulgaris and pemphigus foliaceus antigens, respectively. Abnormal expression of E-cadherin and cytoplasmic plaque proteins such as desmoplakin and plakoglobin was also observed. Western blotting study demonstrated that three squamous cell carcinoma cell lines expressed two desmogleins with a predominant 150 kDa molecule, and a minor 130 kDa one. Although these molecular sizes were similar to those of cultured normal human keratinocytes, the 130 kDa desmoglein, which usually carries pemphigus antigenic epitopes, was weakly or negatively reactive with pemphigus vulgaris serum. One squamous cell carcinoma cell line showed a doublet of 140 and 145 kDa bands in addition to the 130 kDa band. All the carcinoma cell lines constantly expressed desmoglein 2 and desmoglein 3 mRNA, whereas cultured normal human keratinocytes always expressed desmoglein 1 and desmoglein 3 mRNA, with or without desmoglein 2 mRNA. These findings indicate that the squamous cell carcinoma cells revealed abnormal expression of desmoglein isoforms, which may be related to tumor cell kinetics such as cell invasion and metastasis.

Antigens↗

PubMed1995

Use of the minimal function for partial structure development in direct methods.

The shake-and-bake procedure, which is based on the minimal function, has been tested and shown to be extremely effective in molecular-fragment recycling applications. Correctly positioned fragments as small as 5% of the scattering power of the structure typically have a 50% chance of producing a solution in a single recycling trial. While starting models for tangent-formula recycling methods normally require an average r.m.s. displacement error of less than approximately 0.25 A from the refined structure to ensure an adequate chance of success, the shake-and-bake method often tolerates r.m.s. model errors well in excess of 0.5 A. Tests indicate that the new method can outperform traditional tangent-formula procedures in difficult structural applications involving multiple copies of pseudosymmetrically related molecules or low-resolution data.

Cholesterol Esters↗

PubMed1994

A cation binding motif stabilizes the compound I radical of cytochrome c peroxidase.

Cytochrome c peroxidase reacts with peroxide to form compound I, which contains an oxyferryl heme and an indolyl radical at Trp-191. The indolyl free radical has a half-life of several hours at room temperature, and this remarkable stability is essential for the catalytic function of cytochrome c peroxidase. To probe the protein environment that stabilizes the compound I radical, we used site-directed mutagenesis to replace Trp-191 with Gly or Gln. Crystal structures of these mutants revealed a monovalent cation binding site in the cavity formerly occupied by the side chain of Trp-191. Comparison of this site with those found in other known cation binding enzymes shows that the Trp-191 side chain resides in a consensus K+ binding site. Electrostatic potential calculations indicate that the cation binding site is created by partial negative charges at the backbone carbonyl oxygen atoms of residues 175 and 177, the carboxyl end of a long alpha-helix (residues 165-175), the heme propionates, and the carboxylate side chain of Asp-235. These features create a negative potential that envelops the side chain of Trp-191; the calculated free energy change for cation binding in this site is -27 kcal/mol (1 cal = 4.184J). This is more than sufficient to account for the stability of the Trp-191 radical, which our estimates suggest is stabilized by 7.8 kcal/mol relative to a Trp radical in solution.

Binding Sites↗

PubMed1994

Role of methionine 230 in intramolecular electron transfer between the oxyferryl heme and tryptophan 191 in cytochrome c peroxidase compound II.

The kinetics of electron transfer from cytochrome c (CC) to yeast cytochrome c peroxidase (CcP) compound I were studied by flash photolysis and stopped-flow spectroscopy. Flash photolysis studies employed horse CC derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis-(bipyridine)ruthenium (Ru-CC). Initial electron transfer from Ru-CC reduced the indole radical on Trp-191 of CcP compound I [CMPI(IV,R.)], producing CMPII(IV,R). This reaction was biphasic for each of several Ru-CC derivatives, with rate constants which varied according to the position of the Ru label. For Ru-27-CC labeled at lysine 27, rate constants of 43,000 and 1600 s-1 were observed at pH 5.0 in 2 mM acetate. After reduction of the indole radical by Ru-CC, intramolecular electron transfer from Trp-191 to the oxyferryl heme in CMPII(IV,R) was observed, producing CMPII(III,R.). The rate constant and extent of this intramolecular electron transfer reaction were independent of both the protein concentration and the Ru-CC derivative employed. The rate constant decreased from 1100 s-1 at pH 5 to 550 s-1 at pH 6, while the extent of conversion of CMPII(IV,R) to CMPII(III,R.) decreased from 56% at pH 5 to 29% at pH 6. The reaction was not detected at pH 7.0 and above. The pH dependence of the rate and extent of this internal electron transfer reaction paralleled the pH dependence of the rate of bimolecular reduction of CMPII(IV,R) by native horse CC measured by stopped-flow spectroscopy at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

The crystal structure of 1,2-dipalmitoyl-sn-glycerol at 123 K.

At 123 K, the crystal structure of 1,2-dipalmitoyl-sn-glycerol (C35H68O5, M(r) = 568.9) is monoclinic with space group P2(1), a = 5.480(1), b = 7.301(1), c = 43.145(7) A, beta = 92.91(1) degrees, V = 1724 A3, Dc = 1.0960 g cm-3 and Z = 2. Integrated X-ray intensities for 3574 independent reflections were measured with Ni-filtered Cu-K alpha radiation (lambda = 1.5418 A, mu = 0.56 mm-1), and these were all used in a full-matrix least-squares refinement which gave R(F2) = 0.142. Bond lengths and angles have e.s.d.s less than 0.01 A and 0.1 degrees for non-hydrogen atoms. Comparison with a previously reported study of the room temperature structure shows a very similar orthorhombic mode of chain packing but significant conformational differences in the polar headgroup. These changes may occur in conjunction with small changes in enthalpy (less than 1 kJ/mol) observed at 202, 193 and 136 K and with a discontinuity in the unit cell volume observed in the range 200-190 K. Analysis of the atomic mean square displacements at 123 K indicates the presence of internal torsional vibrations in the headgroup region that involve the carbonyl and hydroxyl O-atoms.

Cold Temperature↗

PubMed1994

Crystal structure of cholesteryl butanoate at 123 K.

Cholesteryl butanoate has a complex crystal structure that differs from those of the three main structure type for cholesteryl esters. It contains four molecules (C31H52O2) unrelated by crystal symmetry. The molecules are packed in almost planar sheets and have molecular long axes nearly parallel. However, the molecules have different orientations about their long axes and furthermore, in a given sheet, one of the independent molecules is antiparallel to the other three. Viewed down the molecular long axes, each molecule has six nearest neighbors, but the detailed environment is different for the four independent molecules. Thus the molecular arrangement has features that are characteristic of the short-range order present in the cholesteric mesophase. The monotropic transformation from the crystalline to the cholesteric phase occurs at 98 degrees C. The crystal structure has been accurately determined using 12,146 independent X-ray reflections having sin theta/lambda < 0.63 A-1. All hydrogen atoms were located from a difference Fourier and were included in a refinement that gave R(F2) = 0.064. The C-C bond lengths have sigma = 0.003 A and C-C-C bond angles have sigma = 0.2 degrees. Conformations for the steroid ring system are similar but there are differences in the C17 side chains and the butanoate chains of the four independent molecules. Analysis of atomic m.s. displacement tensors using a segmented-body model indicates that there are internal librations involving both the C17 and butanoate chains in all molecules.

Cholesterol Esters↗

PubMed1991

Crystal structure of cholestanyl caprylate and binary phase behavior with cholesteryl caprylate.

The crystal structure of cholestanyl n-octanoate (caprylate) (C35H62O2) is monoclinic with space group A2 and cell dimensions a = 10.103(7), b = 7.646(7), c = 87.63(7) A, beta = 90.51(6) degrees; Z = 8 [two molecules (A, B) in asymmetric unit], V = 6769 A3, Dc = 1.010 g cm-3. Integrated X-ray intensities for 3798 reflections with I greater than 2 sigma (I) were measured with a rotating anode diffractometer at room temperature. The structure was determined using direct methods. Block diagonal least squares refinement gave R = 0.111. Molecules A and B have almost fully extended conformations, but differ significantly in the rotation about the ester bond and in the C17 chains. The molecular packing in the crystal structure of cholestanyl caprylate consists of stacked bilayers each having d002 = 43.8 A in thickness and within each bilayer, cholestanols pack with cholestanols and caprylate chains pack with caprylate chains. The crystal structure is very similar to that of cholesteryl myristate but is quite different from that of cholesteryl caprylate. The phase equilibria of the cholestanyl caprylate/cholesteryl caprylate binary system have been shown to involve limited mutual solubility of the two components and to have a eutectic point at 73% cholestanyl caprylate. The cholesteric mesophase is monotropic at all compositions except for a narrow range near the eutectic point where it is enantiotropic.

Cholesterol Esters↗