Amide III SFG Signals as a Sensitive Probe of Protein Folding at Cell Membrane Surface

Huang, Jiahuang; Tian, Ke; Ye, Shuji; Luo, Yi

doi:10.1021/acs.jpcc.6b05677

Cited by 25 publications

(45 citation statements)

References 62 publications

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“…To discern them, we further measured the amide III signals. It has been verified that the SFG amide III signals are capable of directly distinguishing α-helix, loop, coil, and turn-like structures in proteins at interfaces. ,− The spectral profiles of the loop and α-helical structures were well separated in the amide III spectra which appeared below and above 1260 cm –1 , respectively . In contrast, the amide III signals of coil and turn-like structures were very weak.…”

Section: Resultsmentioning

confidence: 87%

“…However, this expectation is opposite to what we have observed. Earlier studies indicated that for the α-helical structure, even if the amide I signal is much weaker than the intensity of the 1660 cm –1 peak (as shown in Figure A at 1 min), the amide III signals can still be detected. ,− For example, in Figure B, even if the amide I signal of GP41 in the POPG bilayer at 1660 cm –1 is 5 times weaker than that of hIAPP in the DPPG bilayer, the amide III signal at 1290 cm –1 can be clearly detected for GP41; this is because the GP41 aggregation involves α-helical intermediates . In addition, this detected signal is also supported by the controlled experiments on the interaction between rat-IAPP and the DPPG lipid bilayer.…”

Section: Resultsmentioning

confidence: 89%

“…With increasing time, the intensity increased and the spectra were dominated by the peaks at 1220 and 1240 cm –1 , which were assigned to β-sheet or loop structures. No signals above 1260 cm –1 were observed, indicating that no α-helical structures formed. ,− Therefore, the 1660 cm –1 peak arose from coil and turn-like structures, excluding the generation of transiently α-helical intermediates during the IAPP oligomerization. It needs to be noted that the absence of the signals above 1260 cm –1 in Figure A is not due to the lack of detection sensitivity or because the peptide adopted an unfavorable orientation.…”

Section: Resultsmentioning

confidence: 97%

See 2 more Smart Citations

Misfolding of a Human Islet Amyloid Polypeptide at the Lipid Membrane Populates through β-Sheet Conformers without Involving α-Helical Intermediates

et al. 2019

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Amyloid formation has been implicated in many fatal diseases, but its mechanism remains to be clarified due to a lack of effective methods that can capture the transient intermediates. Here, we experimentally demonstrate that sum frequency generation vibrational spectroscopy can unambiguously discriminate the intermediates during amyloid formation at the lipid membrane in situ and in real time by combining the chiral amide I and achiral amide II and amide III spectral signals of the protein backbone. Such a combination can directly identify the formation of β-hairpin-like monomers and β-sheet oligomers and fibrils. A strong correlation between the amide II signals and the formation of β-sheet oligomers and fibrils was found. With this approach, the structural evolution of human islet amyloid polypeptides (hIAPP) at negative lipid bilayers was elucidated. It was firmly confirmed that hIAPP populates through β-sheet conformers without involving α-helical intermediates. The membrane-associated assembly of hIAPP proceeds by assembling with a β-hairpin-like monomer at the lipid bilayer surface, rather than by inserting the preassembled β-sheet oligomers in solution. This newly established protocol is ready to be utilized in revealing the mechanism of amyloid aggregation at the lipid membrane.

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 97%

See 1 more Smart Citation

Misfolding of a Human Islet Amyloid Polypeptide at the Lipid Membrane Populates through β-Sheet Conformers without Involving α-Helical Intermediates

et al. 2019

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“…SFG vibrational spectroscopy as a surface sensitive technique has been widely used to study chemical and biological molecules on the surface or interface. − SFG theory − and experimental details − have been extensively reported and will not be repeated here. In this study, SFG experiments were performed using a commercial SFG system from EKSPLA.…”

Section: Experimental Sectionmentioning

confidence: 99%

Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity

et al. 2018

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Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitroreductase (NfsB), to demonstrate that engineered variants with two strategically positioned surface-tethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified, and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.

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“…1). The prism-cleaning and lipid monolayer/bilayer preparations were performed using a standard procedure given previously [31][32][33][34].…”

Section: A Materials and Sample Preparationsmentioning

confidence: 99%

Real-Time observation of protein transport across membranes by femtosecond sum frequency generation vibrational spectroscopy

Tan

Zhang

et al. 2018

Chinese Journal of Chemical Physics

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Characterization of conformation kinetics of proteins at the interfaces is crucial for understanding the biomolecular functions and the mechanisms of interfacial biological action. But it requires to capture the dynamic structures of proteins at the interfaces with sufficient structural and temporal resolutions. Here, we demonstrate that a femtosecond sum frequency generation vibrational spectroscopy (SFG-VS) system developed by our group provides a powerful tool for monitoring the real-time peptide transport across the membranes with time resolution of less than one second. By probing the real-time SFG signals in the amide I and amide A bands as WALP23 interacts with DMPG lipid bilayer, it is found that WALP23 is initially absorbed at the gel-phase DMPG bilayer with a random coil structure. The absorption of WALP23 on the surface leads to the surface charge reversal and thus changes the orientation of membrane-bound water. As the DMPG bilayer changes from gel phase into fluid phase, WALP23 inserts into the fluid-phase bilayer with its N-terminal end moving across the membrane, which causes the membrane dehydration and the transition of WALP23 conformation from random coil to mixed helix/loop structure and then to pure α-helical structure. The established system is ready to be employed in characterizing other interfacial fast processes, which will be certainly helpful for providing a clear physical picture of the interfacial phenomena.

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Amide III SFG Signals as a Sensitive Probe of Protein Folding at Cell Membrane Surface

Cited by 25 publications

References 62 publications

Misfolding of a Human Islet Amyloid Polypeptide at the Lipid Membrane Populates through β-Sheet Conformers without Involving α-Helical Intermediates

Misfolding of a Human Islet Amyloid Polypeptide at the Lipid Membrane Populates through β-Sheet Conformers without Involving α-Helical Intermediates

Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity

Real-Time observation of protein transport across membranes by femtosecond sum frequency generation vibrational spectroscopy

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