跳到主要內容
 
:::

余慈顏 博士 研究成果

核磁共振光譜學實驗室
電 子  郵 件:點此顯示(開新頁)
辦    公    室:R231
辦公室電話:+886-2-2366-8210
實    驗    室:221實驗室
實驗室電話:
跳至第 [ 1 ] [ 2 ] 頁
第1頁


 Peibin Zhong,# Chun Hao Liu,# Yit-Tsong Chen* and Tsyr-Yan Yu*
ACS Appl. Bio Mater. 2020, 3, 9, 6351–6357; DOI: 10.1021/acsabm.0c00783
The study of HIV-1 Vpr-membrane and Vpr-hVDAC-1 interactions by Graphene Field-Effect Transistor Biosensors
The viral protein R (Vpr) of human immunodeficiency virus 1 (HIV-1) is involved in many cellular processes during the viral life cycle; however, its associated mechanisms remain unclear. Here, we designed an E. coli expression construct to achieve a milligram yield of recombinant Vpr. In addition, we fabricated a graphene field-effect transistor (G-FET) biosensor, with the modification of a supported lipid bilayer (SLB), to study the interaction between Vpr and its interaction partners. The Dirac point of the SLB/G-FET was observed to shift in response to the binding of Vpr to SLB. By fitting the normalized shift of the Dirac point as a function of Vpr concentration to the Langmuir adsorption isotherm equation, we could extract the dissociation constant (Kd) to quantify the Vpr-binding affinity. When 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) mem-brane was used as the SLB, the dissociation constant was determined to be 9.6 ± 2.1 μM. In contrast, only a slight shift of the Dirac point was observed in response to the addition of Vpr when 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane was used as the SLB. Taking the advantage of the much weaker binding of Vpr to DOPC membrane, we prepared human voltage-dependent anion channel isoform 1 (hVDAC-1) embed-ded DOPC membrane as the SLB for G-FET, and used it to determine the dissociation constant as 5.1 ± 0.9 μM. In summary, using clinically relevant Vpr protein as an example, we demonstrated that a SLB/G-FET bio-sensor is a suitable tool for studying the interaction between a membrane associated protein and its interac-tion partners.
Yo-Tsen Liu, Yueh-Jung Yen, Frans Ricardo, Yu Chang, Pei-Hao Wu, Shing-Jong Huang, Kon-Ping Lin*,Tsyr-Yan Yu*
Annals of Clinical and Translational Neurology, 2019 Oct;6(10):1961-1970. DOI:10.1002/acn3.50887
Biophysical characterization and modulation of Transthyretin Ala97Ser
Objective: Ala97Ser (A97S) is the major transthyretin (TTR) mutation in Taiwanese patients of familial amyloid polyneuropathy (FAP), characterized by a late-onset but rapidly deteriorated neuropathy. Tafamidis can restore the stability of some mutant TTR tetramers and slow down the progression of TTR-FAP. However, there is little understanding of the biophysical features of A97S-TTR mutant and the pharmacological modulation effect of tafamidis on it. This study aims to delineate the biophysical characteristics of A97S-TTR and the pharmacological modulation effect of tafamidis on this mutant.
Method: The stability of TTR tetramers was assessed by urea denaturation and differential scanning calorimetry. Isothermal titration calorimetry (ITC) was used to measure the binding constant of tafamidis to TTR. Nuclear magnetic resonance spectroscopy (NMR) titration experiment was used to map out the tafamidis binding site.
Results: Chemical and thermal denaturation confirmed the destabilization effect of A97S. In consistent with other amyloidogenic mutant, A97S-TTR has slightly lower conformational stability. NMR revealed the binding site of A97S-TTR with tafamidis is at the thyroxine binding pocket. The ITC experiments documented the high affinity of the binding which can effectively stabilize the A97S-TTR tetramer.
Interpretation: This study confirmed the structural modulation effect of tafamidis on A97S-TTR and implied the potential therapeutic benefit of tafamidis for A97S TTR-FAP. This approach can be applied to investigate the modulation effect of tafamidis on other rare TTR variants and help to make individualized choices of available treatments for FAP patients.
Matthew T. Eddy, Tsyr-yan Yu, Gerhard Wagner, Robert G. Griffin
Journal of Biomolecular NMR DOI: 10.1007/s10858-019-00242-8
The second isoform of the human voltage dependent anion channel (VDAC2) is a mitochondrial porin that translocates calcium and other metabolites across the outer mitochondrial membrane. VDAC2 has been implicated in cardioprotection and plays a critical role in a unique apoptotic pathway in tumor cells. Despite its medical importance, there have been few biophysical studies of VDAC2 in large part due to the difficulty of obtaining homogeneous preparations of the protein for spectroscopic characterization. Here we present high resolution magic angle spinning nuclear magnetic resonance (NMR) data obtained from homogeneous preparation of human VDAC2 in 2D crystalline lipid bilayers. The excellent resolution in the spectra permit several sequence-specific assignments of the signals for a large portion of the VDAC2 N-terminus and several other residues in two- and three-dimensional heteronuclear correlation experiments. The first 12 residues appear to be dynamic, are not visible in cross polarization experiments, and they are not sufficiently mobile on very fast timescales to be visible in 13C INEPT experiments. A comparison of the NMR spectra of VDAC2 and VDAC1 obtained from highly similar preparations demonstrates that the spectral quality, line shapes and peak dispersion exhibited by the two proteins are nearly identical. This suggests an overall similar dynamic behavior and conformational homogeneity, which is in contrast to two earlier reports that suggested an inherent conformational heterogeneity of VDAC2 in membranes. The current data suggest that the sample preparation and spectroscopic methods are likely applicable to studying other human membrane porins, including human VDAC3, which has not yet been structurally characterized
Yu-Min KaoChuan-Hao ChengMing-Lun SyueHsin-Yu HuangI-Chia ChenTsyr-Yan Yu*, and Li-Kang Chu*
J. Phys. Chem. B
Photochemistry of Bacteriorhodopsin with Various Oligomeric Statuses in Controlled Membrane Mimicking Environments: A Spectroscopic Study from Femtoseconds to Milliseconds
Preparing transmembrane protein in controllable lipid bilayers is essential for unravelling the coupling of the environments and its dynamic functions. Monomerized bacteriorhodopsin (mbR) embedded in covalently circularized nanodiscs was prepared with dimyristoyl phosphatidylglycerol (DMPG) lipid and circular membrane scaffold proteins of two different sizes, cE3D1 and cΔH5, respectively. The retinal photoisomerization kinetics and thermodynamic photocycle were examined by femtosecond and nanosecond transient absorption, respectively, covering the time scale from femtoseconds to hundreds of milliseconds. The kinetics of the retinal isomerization and proton migration from the protonated Schiff base to Asp-85 were not significantly different for monomeric bR solubilized in Triton X-100 or embedded in circularized nanodiscs. This can be ascribed to the local tertiary structures at the retinal pocket vicinity being similar among monomeric bR in various membrane mimicking environments. However, the aforementioned processes are intrinsically different for trimeric bR in purple membrane (PM) and delipidated PM. The reprotonation of the deprotonated Schiff base from Asp-96 in association with the decay of intermediate M, which involved wide-ranged structural alteration, manifested a difference in terms of the oligomeric statuses, as well as a slight dependence on the size of the nanodisc. In summary, bR oligomeric statuses, rather than the environmental factors, such as membrane mimicking systems and nanodisc size, play a significant role in bR photocycle associated with short-range processes, such as the retinal isomerization and deprotonation of protonated Schiff base at the retinal pocket. On the other hand, the environmental factors, such as the types of membrane mimicking systems and the size of nanodiscs, affect those dynamic processes involving wider structural alterations during the photocycle.
Vivien Yeh, Tsung-Yen Lee, Chung-Wen Chen, Pai-Chia Kuo, Jessie Shiue, Li-Kang Chu* & Tsyr-Yan Yu*
Scientific Reports, 8, 13501 (2018).
Highly Efficient Transfer of 7TM Membrane Protein from Native Membrane to Covalently Circularized Nanodisc
Incorporating membrane proteins into membrane mimicking systems is an essential process for biophysical studies and structure determination. Monodisperse lipid nanodiscs have been found to be a suitable tool, as they provide a near-native lipid bilayer environment. Recently, a covalently circularized nanodisc (cND) assembled with a membrane scaffold protein (MSP) in circular form, instead of conventional linear form, has emerged. Covalently circularized nanodiscs have been shown to have improved stability, however the optimal strategies for the incorporation of membrane proteins, as well as the physicochemical properties of the membrane protein embedded in the cND, have not been studied. Bacteriorhodopsin (bR) is a seven-transmembrane helix (7TM) membrane protein, and it forms a two dimensional crystal consisting of trimeric bR on the purple membrane of halophilic archea. Here it is reported that the bR trimer in its active form can be directly incorporated into a cND from its native purple membrane. Furthermore, the assembly conditions of the native purple membrane nanodisc (PMND) were optimized to achieve homogeneity and high yield using a high sodium chloride concentration. Additionally, the native PMND was demonstrated to have the ability to assemble over a range of different pHs, suggesting flexibility in the preparation conditions. The native PMND was then found to not only preserve the trimeric structure of bR and most of the native lipids in the PM, but also maintained the photocycle function of bR. This suggests a promising potential for assembling a cND with a 7TM membrane protein, extracted directly from its native membrane environment, while preserving the protein conformation and lipid composition.
Orion Shih, Yi-Qi Yeh, Kuei-Fen Liao, Chun-Jen Su, Pei-Hao Wu, Richard K. Heenan, Tsyr-Yan Yu*, and U-Ser Jeng*
Journal of Physical Chemistry Letters
Membrane Charging and Swelling upon Calcium Adsorption as Revealed by Phospholipid Nanodiscs
Direct binding of calcium ions (Ca2+) to phospholipid membranes is an unclarified yet critical signaling pathway in diverse Ca2+-regulated cellular phenomena. Here, high-pressure-liquid-chromatography, small-angle X-ray scattering (SAXS), UV-Vis absorption, and differential refractive index detections are integrated to probe Ca2+-binding to the zwitterionic lipid membranes in nanodiscs. The responses of the membranes upon Ca2+-binding, in composition and conformation, are quantified through integrated data analysis. The results indicate that Ca2+ binds specifically into the phospholipid head-group zone, resulting in membrane charging and membrane swelling, with a saturated Ca2+-lipid binding ratio of 1:8.  A Ca2+-binding isotherm to the nanodisc is further established and yields an unexpectedly high binding constant K = 4260 M-1 and a leaflet potential of ca. 100 mV based on a modified Gouy-Chapman model. The calcium-lipid binding ratio, however, drops to 40% when the nanodisc undergoes a gel-to-fluid phase transition, leading to an effective charge capacity of a few mF/cm2.
 
目前位置:本所人員 / 研究人員 / 余慈顏 / 全部研究成果
回到最上層