Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 973 records · Page 54Linked to original sources

PubMed1999

Synthesis, characterization and the effect of ligand planarity of [Ru(bpy)2L]2+ on DNA binding affinity.

Two structurally related ligands (L) 4,5,9,18-tetraazaphenanthreno[9,10-b] triphenylene (taptp) and 2,3-diphenyl-1,4,8,9-tetraazatriphenylene (dptatp), and their related complexes of [Ru(bpy)2L]2+ have been synthesized and characterized by elemental analyses, 1H NMR and mass spectra. Their electrochemical properties were also examined. Both complexes emit intense luminescence in organic solvent but are quenched in water to different extents. The interactions of the complexes with calf thymus DNA have been investigated by viscosity, absorption, emission and circular dichroism spectra. The intrinsic binding constants of [Ru(bpy)2(taptp)]2+ and [Ru(bpy)2(dptatp)]2+ are 1.7 x 10(5) and 3.8 x 10(4) M-1, respectively. All data indicate that both complexes bind enantioselectively to double-stranded calf thymus DNA via the intercalative mode, with stronger affinity for the fully planar ligand complex of [Ru(bpy)2(taptp)]2+.

2,2'-Dipyridyl↗

PubMed1999

Glucocorticoid regulation of cardiac K+ currents and L-type Ca2+ current in neonatal mice.

Previous studies have reported that dexamethasone (Dex) prolongs cardiac action potential repolarization in mice and rats. However, the cellular mechanisms of this effect have not been addressed. Because action potential duration is influenced by a complex interplay of both inward and outward currents, this study evaluated the role of K+ currents and the L-type Ca2+ current in response to chronic in vivo Dex treatment. Accordingly, neonatal mice were randomly allocated to treatment with Dex (1 mg/kg per day) or placebo (saline) given subcutaneously for 5 days. At 14 to 15 days of age, the L-type Ca2+ current and K+ currents were recorded in ventricular myocytes using whole-cell patch-clamp techniques. The density of peak outward K+ currents was significantly decreased in the chronic Dex-treated group, but the current measured at the end of a 1-second depolarization pulse was similar in both groups. We further measured the magnitudes of the fast-inactivating (I(to)) and the slowly inactivating (I(slow)) currents that contribute to the peak outward K+ currents. I(to) was reduced from 17.5+/-3.0 pA/pF (control) to 10.6+/-2.5 pA/pF (Dex) at +50 mV (P<0.05), but I(slow) was not significantly different. These data suggest that downregulation of I(to) is responsible for the reduced peak outward current. Time courses of the onset and offset of in vivo Dex effects were also assessed. A period of 3 days of treatment was required to observe the Dex effect on peak outward K(+) currents, whereas a 7-day period after discontinuation of Dex was required to recover the baseline current density. Acute in vitro treatment with Dex (1 micromol/L) had no effect on K+ current densities. In addition, chronic Dex treatment significantly increased the density of the L-type Ca2+ current (I(Ca-L)) from -7.2+/-0.5 pA/pF of control to -8.9+/-0.6 pA/pF of Dex at +10 mV, P<0.05. In conclusion, chronic in vivo Dex treatment decreases I(to) and increases I(Ca-L) in neonatal mouse ventricular myocytes, both of which contribute to the prolongation of cardiac action potential repolarization induced by glucocorticoids.

Action Potentials↗

PubMed1999

The design and synthesis of ALS inhibitors from pharmacophore models.

In search of new ALS inhibitors without the previous knowledge of receptor crystal structure, DISCO module was applied to produce 3D-pharmacophore models, which provided information to design novel molecules by 3D-database searching. Then a number of molecules were synthesized. Several of them have some ALS inhibitory activities.

Acetolactate Synthase↗

PubMed1999

Identification of pharmacokinetically stable 3, 10-dibromo-8-chlorobenzocycloheptapyridine farnesyl protein transferase inhibitors with potent enzyme and cellular activities.

Farnesyl protein transferase (FPT) is a promising target for the development of cancer chemotherapeutics because it is responsible for the farnesylation of oncogenic p21 Ras proteins which are found in nearly 30% of all human cancers and necessary for cellular development and growth. The recent discovery and progression to phase II clinical trials of trihalobenzocycloheptapyridine Sch-66336 as a potent inhibitor of FPT with oral, in vivo efficacy in mice have spawned extensive structure-activity relationship studies (SAR) of this class of compounds. Of the many trihalobenzocycloheptapyridine analogues prepared, we have identified several which inhibit FPT and cellular proliferation at single-digit nanomolar concentrations and which have good pharmacokinetic properties in mice.

Administration, Oral↗

PubMed1999

Transient effect of platelet-derived growth factor on GLUT4 translocation in 3T3-L1 adipocytes.

We earlier developed a novel method to detect translocation of the glucose transporter (GLUT) directly and simply using c-MYC epitope-tagged GLUT (GLUTMYC). To define the effect of platelet-derived growth factor (PDGF) on glucose transport in 3T3-L1 adipocytes, we investigated the PDGF- and insulin-induced glucose uptake, translocation of glucose transporters, and phosphatidylinositol (PI) 3-kinase activity in 3T3-L1, 3T3-L1GLUT4MYC, and 3T3-L1GLUT1MYC adipocytes. Insulin and PDGF stimulated glucose uptake by 9-10- and 5.5-6.5-fold, respectively, in both 3T3-L1 and 3T3-L1GLUT4MYC adipocytes. Exogenous GLUT4MYC expression led to enhanced PDGF-induced glucose transport. In 3T3-L1GLUT4MYC adipocytes, insulin and PDGF induced an 8- and 5-fold increase in GLUT4MYC translocation, respectively, determined in a cell-surface anti-c-MYC antibody binding assay. This PDGF-induced GLUT4MYC translocation was further demonstrated with fluorescent detection. In contrast, PDGF stimulated a 2-fold increase of GLUT1MYC translocation and 2.5-fold increase of glucose uptake in 3T3-L1GLUT1MYC adipocytes. The PDGF-induced GLUT4MYC translocation, glucose uptake, and PI 3-kinase activity were maximal (100%) at 5-10 min and thereafter rapidly declined to 40, 30, and 12%, respectively, within 60 min, a time when effects of insulin were maximal. Wortmannin (0.1 microM) abolished PDGF-induced GLUT4MYC translocation and glucose uptake in 3T3-L1GLUT4MYC adipocytes. These results suggest that PDGF can transiently trigger the translocation of GLUT4 and stimulate glucose uptake by translocation of both GLUT4 and GLUT1 in a PI 3-kinase-dependent signaling pathway in 3T3-L1 adipocytes.

3T3 Cells↗

PubMed1999

Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A.

The human p300/CBP-associating factor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 A crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N- and C-terminal protein segments. A correlation of the structure with the extensive mutagenesis data for PCAF and the homologous yeast GCN5 protein implicates the cleft and the N- and C-terminal protein segments as playing an important role in histone substrate binding, and a glutamate residue in the protein core as playing an essential catalytic role. A structural comparison with the coenzyme-bound forms of the related N-acetyltransferases, HAT1 (yeast histone acetyltransferase 1) and SmAAT (Serratia marcescens aminoglycoside 3-N-acetyltransferase), suggests the mode of substrate binding and catalysis by these enzymes and establishes a paradigm for understanding the structure-function relationships of other enzymes that acetylate histones and transcriptional regulators to promote activated transcription.

Acetylation↗

PubMed1999

A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor.

Results of binding assays using DNA fork junction probes indicate that sigma 54 contains multiple determinants that regulate melting to allow RNA polymerase to remain in closed promoter complexes in order to respond to enhancers. Gel mobility shift studies indicate that the -12 promoter element and parts of sigma 54 act together to form a molecular switch that controls melting. The DNA sequences and the sigma 54 N-terminus help direct polymerase to the location within the -12 promoter element where melting will initiate. However, the fork junction that would lead to melting does not form, due to the action of an inhibitory DNA element. Such unregulated melting is inhibited further by the lack of availability of the single-strand binding elements, which are needed to spread opening from the junction to the transcription start site. Thus, in the absence of looping enhancer protein, proper regulation is maintained as the sigma 54 polymerase remains bound in an inactive state. These complex protein-DNA interactions allow the controls over protein recruitment and DNA melting to be separated, enhancing the diversity of accessible mechanisms of transcription regulation.

Base Pairing↗

PubMed1999

A requirement for K+-channel activity in growth factor-mediated extracellular signal-regulated kinase activation in human myeloblastic leukemia ML-1 cells.

Voltage-gated K+ channels have been shown to be required for proliferation of various types of cells. Much evidence indicates that K+-channel activity is required for G1 progression of the cell cycle in different cell backgrounds, suggesting that K+-channel activity is required for early-stage cell proliferation in these cells. However, little is known about the molecular mechanisms that underlie this phenomenon. We have shown in human myeloblastic leukemia ML-1 cells that K+ channels are activated by epidermal growth factor (EGF), whereas serum starvation deprivation suppressed their activity. In addition, voltage-gated K+ channels are required for G1/S-phase transition of the cell cycle. We report here that suppression of K+ channels prevented the activation of extracellular signal-regulated protein kinase 2 (ERK-2) in response to EGF and serum. However, blockade of K+ channels did not prevent ERK-2 activation induced by 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Elimination of extracellular Ca2+ did not alter either ERK-2 activation or the effect of K+-channel blockade on ERK-2 activation. Our data demonstrate that the K+ channel is a part of the EGF-mediated mitogenic signal-transduction process and is required for initiation of the EGF-mediated mitogen-activated protein kinase (MAPK) pathways. Our findings may thus explain why an increase in K+-channel activity is associated with cell proliferation in many types of cells, including ML-1 cells.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

DNA damage by copper(II) complexes: coordination-structural dependence of reactivities.

A variety of copper complexes with different structural features have been shown to bind double-helical DNA with binding constants of 10(4)-10(7) M-1 and to promote double-strand DNA damage upon reductant/H2O2 activation. The interaction of the Cu complex with DNA results in hyperchromism and shifts to longer wavelengths of the strongest transitions in the Cu complexes, as well as striking hypochromism or hyperchromism of DNA absorption at 260 nm. In the presence of DMPO as the spin trap, the solution of each copper complex exhibits typical four-line ESR spectra of the hydroxyl radical by adding 2-mercaptoethanol and H2O2 to the solution. Quantitation by 2-deoxy-D-ribose shows that the competence of hydroxyl radical generation by the copper complexes upon reductant and H2O2 activation decreases in order, that is, Cu(HTCD)(2+)-Cu(Im)4Cl2-Cu(IDB) (NO3)2 > Cu(IDB)Cl2 > Cu(IDBt)Cl2. The copper complex-mediated hydroxyl radical, a powerful oxidant that attacks the adjacent DNA, is responsible for the DNA oxidative damage. The lambda DNA damage chemistry illustrates that the competence and selectivity of double-strand lambda DNA damage by the copper complexes are dependent on their geometric structures and types of ligands. The decreasing order of the DNA damage capacity by the present complexes is Cu(Im)4Cl2-Cu(IDB)-(NO3)2 > Cu(HTCD)2+ > Cu(IDBt)Cl2 > Cu(IDB)Cl2.

Animals↗

PubMed1999

Secretory event in intestinal grafts during preservation ischemia.

BACKGROUND: Ischemia triggers secretion of proteins from the intestine, including type II secretory phospholipase A2 (sPLA2). This "secretory event" was studied in intestinal grafts during the first few hours of preservation by measuring total protein, sPLA2, and other enzymes in the UW preservation solution over time. The effect of PX-13, a PLA2 inhibitor, was also studied. MATERIALS AND METHODS: Twenty-five centimeter intestinal grafts were harvested from Lewis rats, flushed, and preserved in UW solution +/- PX-13 at 4 degrees C. UW samples from 0 to 48 h (n = 5 each) were analyzed for total protein, sPLA2, lactate dehydrogenase (LDH), N-acetylglucosamine (NAGA), and lysozyme. Nonpreserved grafts were homogenized in PBS as tissue controls. Standard biochemical methods were used for all assays. RESULTS: Total protein increased rapidly by 5 min, continued to rise more slowly until 30 min, and then stabilized. The most significant increase in sPLA2 activity occurred between 90 and 180 min. NAGA increased most markedly between 30 and 180 min, while LDH increased in the first 30 min, although the level of both enzymes was negligible compared to tissue enzyme. Lysozyme levels were minimal at all times. PX-13 decreased sPLA2 activity markedly at all time points. CONCLUSION: Total protein levels increased before sPLA2, suggesting that sPLA2 may be secreted in response to other proteins or enzymes released even earlier during preservation (e.g., cytokines). These elevations do not appear to be caused by cell death. Phospholipase A2 secretion may be blocked, and this may greatly improve the outcome of intestinal preservation.

Acetylglucosamine↗

PubMed1999

p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells.

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.

Adult↗

PubMed1999

Induction of MHC class I expression on immature thymocytes in HIV-1-infected SCID-hu Thy/Liv mice: evidence of indirect mechanisms.

The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.

Animals↗

PubMed1999

Carboxypeptidase A3 (CPA3): a novel gene highly induced by histone deacetylase inhibitors during differentiation of prostate epithelial cancer cells.

Butyrate and its structural analogues have recently entered clinical trials as a potential drug for differentiation therapy of advanced prostate cancer. To better understand the molecular mechanism(s) involved in prostate cancer differentiation, we used mRNA differential display to identify the gene(s) induced by butyrate. We found that the androgen-independent prostate cancer cell line PC-3 undergoes terminal differentiation and apoptosis after treatment with sodium butyrate (NaBu). A novel cDNA designated carboxypeptidase A3 (CPA3), which was up-regulated in NaBu-treated PC-3 cells, was identified and characterized. This gene expresses a 2795-bp mRNA encoding a protein with an open reading frame of 421 amino acids. CPA3 has 37-63% amino acid identity with zinc CPs from different mammalian species. It also shares 27-43% amino acid similarity with zinc CPs from several nonmammalian species, including Escherichia coli, yeast, Caenorhabditis elegans, and Drosophila. The structural similarity between CPA3 and its closest homologues indicates that the putative CPA3 protein contains a 16-residue signal peptide sequence, a 95-residue NH2-terminal activation segment, and a 310-residue CP enzyme domain. The consistent induction of CPA3 by NaBu in several prostate cancer cell lines led us to investigate the signaling pathway involved in the induction of CPA3 mRNA. Trichostatin A, a potent and specific inhibitor of histone deacetylase, also induced CPA3 mRNA expression, suggesting that CPA3 gene induction is mediated by histone hyperacetylation. We demonstrated that CPA3 induction was a downstream effect of the treatment with butyrate or trichostatin A, but that the induction of p21(WAF1/CIP1) occurred immediately after these treatments. We also demonstrated that the induction of CPA3 mRNA by NaBu was inhibited by p21(WAF1/CIP1) antisense mRNA expression, indicating that p21 transactivation is required for the induction of CPA3 by NaBu. Our data demonstrate that the histone hyperacetylation signaling pathway is activated during NaBu-mediated differentiation of PC-3 cells, and the new gene, CPA3, is involved in this pathway.

Acetylation↗

PubMed1999

Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme.

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262-4266) and is known as the "10-23" general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3'-3'-internucleotide inversion at the 3'-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity.

Animals↗

PubMed1999

Bacterial expression of the major antigenic regions of porcine rotavirus VP7 induces a neutralizing immune response in mice.

The outer capsid protein of rotavirus, VP7, is a major neutralization antigen. A chimeric protein comprising Escherichia coli (E. coli) outer membrane protein A (OmpA) and part of porcine rotavirus VP7 containing all three antigenic regions (217 amino acids) was expressed in Salmonella and E. coli as an outer-membrane associated protein. Mice immunized intraperitoneally or orally, respectively, with live E. coli or Salmonella cells expressing this chimeric protein produced antibodies against native VP7 as determined by enzyme-linked immunosorbent assays and neutralization tests. This indicates that the VP7 fragment from a porcine rotavirus which is antigenically similar to human rotavirus serotype 3, when expressed in bacteria as a chimeric protein, can form a structure resembling its native form at least in some of the major neutralization domains. These results indicate that the use of a live bacterial vector expressing rotavirus VP7 may represent a strategy for the development of vaccines against rotavirus-induced diarrhoea in infants.

Animals↗

PubMed1999

NH2-Terminal targeting motifs direct dual specificity A-kinase-anchoring protein 1 (D-AKAP1) to either mitochondria or endoplasmic reticulum.

Subcellular localization directed by specific targeting motifs is an emerging theme for regulating signal transduction pathways. For cAMP-dependent protein kinase (PKA), this is achieved primarily by its association with A-kinase-anchoring proteins (AKAPs). Dual specificity AKAP1, (D-AKAP1) binds to both type I and type II regulatory subunits and has two NH2-terminal (N0 and N1) and two COOH-terminal (C1 and C2) splice variants (. J. Biol. Chem. 272:8057). Here we report that the splice variants of D-AKAP1 are expressed in a tissue-specific manner with the NH2-terminal motifs serving as switches to localize D-AKAP1 at different sites. Northern blots showed that the N1 splice is expressed primarily in liver, while the C1 splice is predominant in testis. The C2 splice shows a general expression pattern. Microinjecting expression constructs of D-AKAP1(N0) epitope-tagged at either the NH2 or the COOH terminus showed their localization to the mitochondria based on immunocytochemistry. Deletion of N0(1-30) abolished mitochondrial targeting while N0(1-30)-GFP localized to mitochondria. Residues 1-30 of N0 are therefore necessary and sufficient for mitochondria targeting. Addition of the 33 residues of N1 targets D-AKAP1 to the ER and residues 1-63 fused to GFP are necessary and sufficient for ER targeting. Residues 14-33 of N1 are especially important for targeting to ER; however, residues 1-33 alone fused to GFP gave a diffuse distribution. N1(14-33) thus serves two functions: (a) it suppresses the mitochondrial-targeting motif located within residues 1-30 of N0 and (b) it exposes an ER-targeting motif that is at least partially contained within the N0(1-30) motif. This represents the first example of a differentially targeted AKAP and adds an additional level of complexity to the PKA signaling network.

A Kinase Anchor Proteins↗

PubMed1999

Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF-RARalpha and NPM-RARalpha.

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation involving RARalpha and one of four fusion partners: PML, PLZF, NPM, and NuMA genes. To study the leukemogenic potential of the fusion genes in vivo, we generated transgenic mice with PLZF-RARalpha and NPM-RARalpha. PLZF-RARalpha transgenic animals developed chronic myeloid leukemia-like phenotypes at an early stage of life (within 3 months in five of six mice), whereas three NPM-RARalpha transgenic mice showed a spectrum of phenotypes from typical APL to chronic myeloid leukemia relatively late in life (from 12 to 15 months). In contrast to bone marrow cells from PLZF-RARalpha transgenic mice, those from NPM-RARalpha transgenic mice could be induced to differentiate by all-trans-retinoic acid (ATRA). We also studied RARE binding properties and interactions between nuclear corepressor SMRT and various fusion proteins in response to ATRA. Dissociation of SMRT from different receptors was observed at ATRA concentrations of 0.01 microM, 0.1 microM, and 1.0 microM for RARalpha-RXRalpha, NPM-RARalpha, and PML-RARalpha, respectively, but not observed for PLZF-RARalpha even in the presence of 10 microM ATRA. We also determined the expression of the tissue factor gene in transgenic mice, which was detected only in bone marrow cells of mice expressing the fusion genes. These data clearly establish the leukemogenic role of PLZF-RARalpha and NPM-RARalpha and the importance of fusion receptor/corepressor interactions in the pathogenesis as well as in determining different clinical phenotypes of APL.

Animals↗

PubMed1999

Helix capping in the GCN4 leucine zipper.

Capping interactions associated with specific sequences at or near the ends of alpha-helices are important determinants of the stability of protein secondary and tertiary structure. We investigate here the role of the helix-capping motif Ser-X-X-Glu, a sequence that occurs frequently at the N termini of alpha helices in proteins, on the conformation and stability of the GCN4 leucine zipper. The 1.8 A resolution crystal structure of the capped molecule reveals distinct conformations, packing geometries and hydrogen-bonding networks at the amino terminus of the two helices in the leucine zipper dimer. The free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is -1.2 kcal/mol, evaluated from thermal unfolding experiments. A single cap thus contributes appreciably to stabilizing the terminated helix and thereby the native state. These results suggest that helix capping plays a further role in protein folding, providing a sensitive connector linking alpha-helix formation to the developing tertiary structure of a protein.

Amino Acid Sequence↗