Jian Liu

Yale University, New Haven, Connecticut, United States

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Publications (14)70.28 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We present measurements of rate constants for thermal-induced reactions of the 11-cis retinyl chromophore in vertebrate visual pigment rhodopsin, a process that produces noise and limits the sensitivity of vision in dim light. At temperatures of 52.0-64.6 °C, the rate constants fit well to an Arrhenius straight line with, however, an unexpectedly large activation energy of 114 ± 8 kcal/mol, which is much larger than the 60-kcal/mol photoactivation energy at 500 nm. Moreover, we obtain an unprecedentedly large prefactor of 10(72±5) s(-1), which is roughly 60 orders of magnitude larger than typical frequencies of molecular motions! At lower temperatures, the measured Arrhenius parameters become more normal: Ea = 22 ± 2 kcal/mol and Apref = 10(9±1) s(-1) in the range of 37.0-44.5 °C. We present a theoretical framework and supporting calculations that attribute this unusual temperature-dependent kinetics of rhodopsin to a lowering of the reaction barrier at higher temperatures due to entropy-driven partial breakup of the rigid hydrogen-bonding network that hinders the reaction at lower temperatures.
    Proceedings of the National Academy of Sciences of the United States of America. 07/2014;
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    ABSTRACT: Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin: thermal isomerization of 11-cis retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37°C and 55°C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark-state hydrolysis of Schiff base by 1-2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen-bond network at the receptor's active site. The decrease in the thermal stability of dark-state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin and potentially with night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin's thermal stability and RP progression in patients.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
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    ABSTRACT: GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca2+ is a weak agonist of PTH1R and Ca2+ in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.
    ACS Chemical Biology 12/2012; · 5.44 Impact Factor
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    ABSTRACT: Emulsions are widely used in industrial and environmental remediation applications. The breaking and reformulation of emulsions, which occur during their use, lead to changes in their surface composition as well as their physical and chemical properties. Hence, a fundamental understanding of the transfer of surfactant molecules between emulsion particles is required for optimization of their applications. However, such an understanding remains elusive because of the lack of in situ and real-time surface-specific techniques. To address this, we designed and synthesized the surfactant probe molecules MG-butyl-1 (2) and MG-octyl-1 (3), which contain an n-butyl and an n-octyl chain, respectively, and a charged headgroup similar to that in malachite green (MG, 1). MG is known to be effective in generating second harmonic generation (SHG) signals when adsorbed onto surfaces of colloidal microparticles. Making use of the coherent nature of SHG, we monitored in real-time the transfer of 2 and 3 between oil-in-water emulsion particles with diameters of ~220 nm. We found that 3 is transferred ~600 times slower than 2, suggesting that an increase in the hydrophobic chain length decreases the transfer rate. Our results show that SHG combined with molecular design and synthesis of surfactant probe molecules can be used to measure the rate of surfactant transfer between emulsion particles. This method provides an experimental framework for examining the factors controlling the kinetics of surfactant transfer between emulsion particles, which cannot be readily investigated in situ and in real-time using conventional methods.
    Journal of the American Chemical Society 03/2012; 134(9):4264-8. · 10.68 Impact Factor
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    ABSTRACT: Many amyloid proteins misfold into β-sheet aggregates upon interacting with biomembranes at the onset of diseases, such as Parkinson's disease and type II diabetes. The molecular mechanisms triggering aggregation depend on the orientation of β-sheets at the cell membranes. However, understanding how β-sheets adsorb onto lipid/aqueous interfaces is challenging. Here, we combine chiral sum frequency generation (SFG) spectroscopy and ab initio quantum chemistry calculations based on a divide-and-conquer strategy to characterize the orientation of human islet amyloid polypeptides (hIAPPs) at lipid/aqueous interfaces. We show that the aggregates bind with β-strands oriented at 48° relative to the interface. This orientation reflects the amphiphilic properties of hIAPP β-sheet aggregates and suggests the potential disruptive effect on membrane integrity.
    Journal of Molecular Biology 12/2011; 421(4-5):537-47. · 3.91 Impact Factor
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    ABSTRACT: The thermal properties of rhodopsin, which set the threshold of our vision, have long been investigated, but the chemical kinetics of the thermal decay of rhodopsin has not been revealed in detail. To understand thermal decay quantitatively, we propose a kinetic model consisting of two pathways: 1) thermal isomerization of 11-cis-retinal followed by hydrolysis of Schiff base (SB) and 2) hydrolysis of SB in dark state rhodopsin followed by opsin-catalyzed isomerization of free 11-cis-retinal. We solve the kinetic model mathematically and use it to analyze kinetic data from four experiments that we designed to assay thermal decay, isomerization, hydrolysis of SB using dark state rhodopsin, and hydrolysis of SB using photoactivated rhodopsin. We apply the model to WT rhodopsin and E181Q and S186A mutants at 55 °C, as well as WT rhodopsin in H(2)O and D(2)O at 59 °C. The results show that the hydrogen-bonding network strongly restrains thermal isomerization but is less important in opsin and activated rhodopsin. Furthermore, the ability to obtain individual rate constants allows comparison of thermal processes under various conditions. Our kinetic model and experiments reveal two unusual energetic properties: the steep temperature dependence of the rates of thermal isomerization and SB hydrolysis in the dark state and a strong deuterium isotope effect on dark state SB hydrolysis. These findings can be applied to study pathogenic rhodopsin mutants and other visual pigments.
    Journal of Biological Chemistry 09/2011; 286(44):38408-16. · 4.65 Impact Factor
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    ABSTRACT: Rhodopsin has developed mechanisms to optimize its sensitivity to light by suppressing dark noise and enhancing quantum yield. We propose that an intramolecular hydrogen-bonding network formed by ∼20 water molecules, the hydrophilic residues, and peptide backbones in the transmembrane region is essential to restrain thermal isomerization, the source of dark noise. We studied the thermal stability of rhodopsin at 55 °C with single point mutations (E181Q and S186A) that perturb the hydrogen-bonding network at the active site. We found that the rate of thermal isomerization increased by 1–2 orders of magnitude in the mutants. Our results illustrate the importance of the intact hydrogen-bonding network for dim-light detection, revealing the functional roles of water molecules in rhodopsin. We also show that thermal isomerization of 11-cis-retinal in solution can be catalyzed by wild-type opsin and that this catalytic property is not affected by the mutations. We characterize the catalytic effect and propose that it is due to steric interactions in the retinal-binding site and increases quantum yield by predetermining the trajectory of photoisomerization. Thus, our studies reveal a balancing act between dark noise and quantum yield, which have opposite effects on the thermal isomerization rate. The acquisition of the hydrogen-bonding network and the tuning of the steric interactions at the retinal-binding site are two important factors in the development of dim-light vision.
    Journal of Biological Chemistry 08/2011; 286(31):27622-27629. · 4.65 Impact Factor
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    ABSTRACT: Rhodopsin has developed mechanisms to optimize its sensitivity to light by suppressing dark noise and enhancing quantum yield. We propose that an intramolecular hydrogen-bonding network formed by ∼20 water molecules, the hydrophilic residues, and peptide backbones in the transmembrane region is essential to restrain thermal isomerization, the source of dark noise. We studied the thermal stability of rhodopsin at 55 °C with single point mutations (E181Q and S186A) that perturb the hydrogen-bonding network at the active site. We found that the rate of thermal isomerization increased by 1-2 orders of magnitude in the mutants. Our results illustrate the importance of the intact hydrogen-bonding network for dim-light detection, revealing the functional roles of water molecules in rhodopsin. We also show that thermal isomerization of 11-cis-retinal in solution can be catalyzed by wild-type opsin and that this catalytic property is not affected by the mutations. We characterize the catalytic effect and propose that it is due to steric interactions in the retinal-binding site and increases quantum yield by predetermining the trajectory of photoisomerization. Thus, our studies reveal a balancing act between dark noise and quantum yield, which have opposite effects on the thermal isomerization rate. The acquisition of the hydrogen-bonding network and the tuning of the steric interactions at the retinal-binding site are two important factors in the development of dim-light vision.
    Journal of Biological Chemistry 06/2011; 286(31):27622-9. · 4.65 Impact Factor
  • Li Fu, Jian Liu, Elsa C Y Yan
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    ABSTRACT: In situ and real-time characterization of protein secondary structures at interfaces is important in biological and bioengineering sciences, yet remains technically challenging. In this study, we used chiral sum frequency generation (SFG) spectroscopy to establish a set of vibrational optical markers for characterizing protein secondary structures at interfaces. We discovered that the N-H stretches along the peptide backbones of α-helices can be detected in chiral SFG spectra. We further observed that the chiral vibrational signatures of the N-H stretch together with the peptide amide I are unique to α-helix, β-sheet, and random coil at interfaces. Using these chiral vibrational signatures, we studied the aggregation of human islet amyloid polypeptide (hIAPP), which is implicated in type II diabetes. We observed in situ and in real time the misfolding of hIAPP from random coils to α-helices and then β-sheets upon interaction with a lipid-water interface. Our findings show that chiral SFG spectroscopy is a powerful tool to follow changes in protein conformations at interfaces and identify interfacial protein secondary structures that elude conventional techniques.
    Journal of the American Chemical Society 06/2011; 133(21):8094-7. · 10.68 Impact Factor
  • Biophysical Journal 02/2011; 100(3):545-. · 3.67 Impact Factor
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    ABSTRACT: The achiral C_(3v) organic phosphine tris(hydroxypropyl)phosphine oxide (1) crystallizes in the unusual chiral hexagonal space group P6_3. The structure is highly ordered because each phosphine oxide moiety forms three hydrogen bonds with adjacent hydroxy groups from three different molecules. The properties of the crystals and the presence of hydrogen bonding interactions were investigated using single crystal Raman spectroscopy. The crystals show nonlinear optical properties and are capable of efficient second harmonic generation.
    Crystal Growth & Design 04/2010; · 4.69 Impact Factor
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    ABSTRACT: Over 120 point mutations in the rhodopsin gene have been associated with the autosomal dominant form of retinitis pigmentosa (RP), a family of heterogeneous inherited visual disorders. We aim to understand the pathogenic mechanism for the mutations S186W and D190N, which are known to perturb an electrostatic interaction and a hydrogen bonding network at the active site. We expressed and purified wild-type (WT) rhodopsin and the mutants from HEK293 cells. Using UV-visible spectroscopy, we measured the rate of thermal decay of the mutants by monitoring the decrease in absorption at 500 nm. No decay was observed for D190N and WT rhodopsin over 24 hours at 37°C, whereas the mutation S186W decayed with a half-life of 36.6 min. We also found that at 55°C the rates of thermal decay for S186W and D190N were 101 and 28 times faster than that for WT rhodopsin, respectively. These results suggest that the mutations thermally destabilize rhodopsin. We also measured the rate of thermal isomerization of 11-cis to all-trans retinal using high-performance liquid chromatography. We found that the mutations increased the rate of thermal isomerization by 1-2 orders of magnitude. Thus, our results indicate that the perturbations to the hydrogen bonding network caused by the D190N and S186W mutations could decrease the thermal stability of rhodopsin, which could be related to the progressive death of rod cells characteristic of RP. The mutations also increase the thermal excitation of rhodopsin, which desensitizes the photoreceptor and potentially leads to the early symptom of night blindness. Our studies provide insight into a molecular understanding of RP and relate the thermal stability of rhodopsin to the rate of progression of the disorder. This could provide more accurate prognoses for RP patients and guide strategies for treatment.
    36th American Chemical Society Northeast Regional Meeting; 10/2009
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    ABSTRACT: Although thermal stability of the G protein-coupled receptor rhodopsin is directly related to its extremely low dark noise level and has recently generated considerable interest, the chemistry behind the thermal decay process of rhodopsin has remained unclear. Using UV-vis spectroscopy and HPLC analysis, we have demonstrated that the thermal decay of rhodopsin involves both hydrolysis of the protonated Schiff base and thermal isomerization of 11-cis to all-trans retinal. Examining the unfolding of rhodopsin by circular dichroism spectroscopy and measuring the rate of thermal isomerization of 11-cis retinal in solution, we conclude that the observed thermal isomerization of 11-cis to all-trans retinal happens when 11-cis retinal is in the binding pocket of rhodopsin. Furthermore, we demonstrate that solvent deuterium isotope effects are involved in the thermal decay process by decreasing the rates of thermal isomerization and hydrolysis, suggesting that the rate-determining step of these processes involves breaking hydrogen bonds. These results provide insight into understanding the critical role of an extensive hydrogen-bonding network on stabilizing the inactive state of rhodopsin and contribute to our current understanding of the low dark noise level of rhodopsin, which enables this specialized protein to function as an extremely sensitive biological light detector. Because similar hydrogen-bonding networks have also been suggested by structural analysis of two other GPCRs, beta1 and beta2 adrenergic receptors, our results could reveal a general role of hydrogen bonds in facilitating GPCR function.
    Journal of the American Chemical Society 08/2009; 131(25):8750-1. · 10.68 Impact Factor
  • Gang Ma, Jian Liu, Li Fu, Elsa C Y Yan
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    ABSTRACT: We have built a broad bandwidth vibrational sum frequency generation (VSFG) spectrometer that can provide high-quality spectra over the range of 3800 to 900 cm(-1). The spectrometer contains a commercial Ti:sapphire based 6 W regenerative amplifier as the master light source, a home-built pulse shaper to produce a narrow bandwidth 800 nm beam, a commercial optical parametric amplifier to generate a broad bandwidth femtosecond infrared (IR) pulse, and a detection system with a monochromator and a charge-coupled device (CCD). We applied this spectrometer to obtain VSFG spectra of a lipid monolayer at the air-water interface in the O-H stretching region (3800-3000 cm(-1)), the C-H stretching region (3100-2700 cm(-1)), the C-D stretching region (2300-2000 cm(-1)), the C=O stretching region (1800-1700 cm(-1)), and the PO(2)(-) symmetric stretching region (1200-1000 cm(-1)). We also obtained the VSFG spectrum of neat water in the O-H stretching region (3800-3000 cm(-1)) and the VSFG spectrum of a protein, alpha-synuclein, in the amide I region (1700-1600 cm(-1)) at the air-water interface. The spectrometer can provide a VSFG spectrum in the O-H stretching region (3800-3000 cm(-1)) without scanning the IR frequency. This feature will be useful in probing water dynamics at interfaces because the free OH and H-bonded OH can be investigated simultaneously. We have also provided instrumental details and discussed further improvements that should be beneficial to other researchers interested in setting up VSFG instrumentation.
    Applied Spectroscopy 06/2009; 63(5):528-37. · 1.94 Impact Factor