Yu-Yung Chang

Interaction of electron and hole plasma with coherent longitudinal optical phonons in GaAs

The interaction of photoexcited electron–hole plasma with impulsively excited coherent longitudinal optical phonons in GaAs was investigated via time-resolved second-harmonic generation. The dephasing time of coherent LO phonons reduces significantly as photoexcited electron–hole plasma is injected into the near-surface depletion region. The coherent LO-electron and LO-hole coupling modes can both be clearly observed and investigated in real time. The rapid dephasing of the coherent LO-hole coupling mode is attributed to the strong polar phonon-carrier interaction and the overdamped nature of hole plasma.

Determination of the electron effective mass of wurtzite InN by coherent upper-branch A1(LO) phonon-plasmon coupling mode

Coherent A1(LO) phonon and its coupling with photoexcited plasmon in wurtzite InN were generated and detected with time-resolved second-harmonic generation. The experimental results directly reveal that the plasma damping time constant is about 60∼120fs, which depends on the photoexcited plasma density in InN. The frequency of the upper-branch A1(LO) phonon-plasmon coupling mode shifts as a function of the photoexcited plasma density. This frequency shift can be fitted consistently with different InN films by solving the InN dielectric response function and leads to the determination of the electron effective mass m‖*=(0.033±0.003)me, parallel to the c axis of wurtzite InN.

Identification and Characterization of an Extracellular Alkaline Phosphatase in the Marine Diatom Phaeodactylum tricornutum

In phosphorus-deficient conditions, Phaeodactylum tricornutum releases an alkaline phosphatase (PtAPase) to the medium that is readily detectable by activity staining. Nucleic acid and amino acid sequence of this alkaline phosphatase (APase) was identified by performing proteomic analysis and database searches. Sequence alignment suggests that PtAPase belongs to the PhoA family, and it possesses key residues at the Escherichia coli PhoA active site. Quantitative PCR results indicate that the induction of APase mRNA transcription is very sensitive to phosphorus availability and population growth. The molecular mass of native PtAPase (148xa0kDa) determined by gel filtration chromatography indicates that PtAPase, like most PhoA, is homodimeric. Zn and Mg ions are essential cofactors for most PhoA enzymes; however, PtAPase activity did not require Zn ions. In fact, 5xa0mM Zn2+, Mo2+, Co2+, Cd2+, or Cu2+ inhibited its enzymatic activity, whereas 5xa0mM Mn2+, Mg2+, or Ca2+ enhanced its enzymatic activity. The responses of PtAPase to divalent metal ions were different from those of most PhoAs, but were similar to the PhoA in a marine bacterium, Cobetia marina. Phylogenetic analysis shows that homologs of PhoA are also present in other diatom species, and that they clustered in a unique branch away from other PhoA members. PtAPase may represent a novel class of PhoA that helps diatoms to survive in the ocean. Quantification of the PtAPase mRNA may help monitor the physiological condition of diatoms in natural environments and artificial bioreactors.

Structural Basis for Substrate-specific Acetylation of Nα-acetyltransferase Ard1 from Sulfolobus solfataricus

Nα-acetyltransferases (Nats) possess a wide range of important biological functions. Their structures can vary according to the first two residues of their substrate. However, the mechanisms of substrate recognition and catalysis of Nats are elusive. Here, we present two structure of Sulfolobus solfataricus Ard1 (SsArd1), a member of the NatA family, at 2.13 and 1.84 Å. Both structures contain coenzyme A, while the latter also contains a substrate-derived peptide. Sequential structure-based mutagenesis revealed that mutations of critical residues for CoA binding decreased the binding affinity of SsArd1 by 3 ~ 7-fold. Superimposition of SsArd1 (NatA) with human Naa50p (NatE) showed significant differences in key residues of enzymes near the first amino-acid position of the substrate peptide (Glu35 for SsArd1 and Val29 for Naa50p). Further enzyme activity assays revealed that the substrate specificity of SsArd1 could be altered from SSGTPT to MEEKVG by a range of Glu35 mutants. These studies provide not only a molecular elucidation of substrate recognition and specificity for the NatA family, but also insight into how members of the NAT family distinguish between amino acids at the substrate N-terminus from the ancient monomeric archaeal Ard1.

Observation of coherent interfacial optical phonons in GaInP/GaAs/GaInP single quantum wells

Coherent phonon spectroscopy of GaInP/GaAs/GaInP single quantum wells is demonstrated with time-resolved second-harmonic generation. Coherent longitudinal optical phonons are impulsively launched via transient pump-induced field screening and stimulated Raman scattering in the well and barrier regions. A phonon mode at 9.4 THz is identified as interfacial phonon localized in the GaAs/GaInP hetero-interface. The free induced dephasing of this coherent interfacial phonon is analyzed with window-gated Fourier transform. Its dephasing time is used to characterize the interface quality of semiconductor heterostructures.

Solution structure of the oncogenic MIEN1 protein reveals a thioredoxin-like fold with a redox-active motif.

The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as −225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.

Structural basis for DNA-mediated allosteric regulation facilitated by the AAA + module of Lon protease

Lon belongs to a unique group of AAA+ proteases that bind DNA. However, the DNA-mediated regulation of Lon remains elusive. Here, the crystal structure of the α subdomain of the Lon protease from Brevibacillus thermoruber (Bt-Lon) is presented, together with biochemical data, and the DNA-binding mode is delineated, showing that Arg518, Arg557 and Arg566 play a crucial role in DNA binding. Electrostatic interactions contributed by arginine residues in the AAA+ module are suggested to be important to DNA binding and allosteric regulation of enzymatic activities. Intriguingly, Arg557, which directly binds DNA in the α subdomain, has a dual role in the negative regulation of ATPase stimulation by DNA and in the domain-domain communication in allosteric regulation of Bt-Lon by substrate. In conclusion, structural and biochemical evidence is provided to show that electrostatic interaction in the AAA+ module is important for DNA binding by Lon and allosteric regulation of its enzymatic activities by DNA and substrate.

Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60°C and specific activity of about 2,600 U/mg with optimal temperature of 90°C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzymes activity.

Using single-molecule approaches to study archaeal DNA-binding protein Alba1.

Thermophilic and hyperthermophilic archaea have one or more copies of the Alba gene, which encodes Alba, a dimeric, highly basic protein that binds cooperatively to DNA. However, the functions of Alba and how it interacts with DNA remain unclear. In this study, we have used single-molecule tethered particle motion (TPM) and optical tweezers (OT) experiments to study the interactions between DNA molecules and Alba1. When Alba1 binds to double-stranded DNA, the Brownian motion (BM) amplitude for DNA tethers increases continuously, suggesting that Alba1 binds cooperatively. The OT study confirmed that a 5-fold increase in the persistence length of the Alba1 nucleoprotein filament is the major factor causing the increase in the BM amplitude for DNA tethers, while the contour length remained mostly unchanged. Moreover, the rate of the increase in the BM amplitude and the BM plateau value are both DNA length-dependent, indicating that the number of Alba1 initiation binding sites increases as the DNA becomes longer. Using the incoming-strand TPM experiment to monitor the interaction between Alba1 nucleoprotein filaments, we found that significant dimer-dimer contacts between two Alba1 nucleoprotein filaments are present, and the interaction is regulated by the concentration of Alba1.

Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus

Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80u2005Å on the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2u2005Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52u2005Å(3)u2005Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.