Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and 13C–2H rotational-echo double-resonance solid-state NMR

Xie, Long; Ghosh, Ujjayini; Schmick, Scott D.; Weliky, David P.

doi:10.1007/s10858-012-9692-8

Cited by 12 publications

(14 citation statements)

References 30 publications

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“…A similar change in ratio with τ is observed for the monomeric α helical KALP peptide in membranes. 6 …”

Section: Resultsmentioning

confidence: 99%

“…4–6 This work is done in the context of a variety of other established approaches to probe residue-specific membrane location. For example, the distance between a Trp indole group and a non-native lipid Br atom can be semi-quantitatively determined from Br-induced quenching of the indole fluorescence.…”

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confidence: 99%

“…5,6,16–18 We were motivated to use an approach for which there was coherent magnetization transfer between spins and where the data analysis could be validated using protein containing isolated spin pairs with a single dipolar coupling d and internuclear distance r with d ∝ r −3 . The buildup of the experimental (Δ S / S 0 ) exp with dephasing time τ is fitted to a long-time extent and buildup rate which are respectively correlated to the fractional population with a particular d .…”

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confidence: 99%

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REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

Jia

Liang

Sackett

et al. 2015

Journal of Magnetic Resonance

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Rotational-echo double-resonance (REDOR) solid-state NMR is applied to probe the membrane locations of specific residues of membrane proteins. Couplings are measured between protein 13CO nuclei and membrane lipid or cholesterol 2H and 31P nuclei. Specific 13CO labeling is used to enable unambiguous assignment and 2H labeling covers a small region of the lipid or cholesterol molecule. The 13CO-31P and 13CO-2H REDOR respectively probe proximity to the membrane headgroup region and proximity to specific insertion depths within the membrane hydrocarbon core. One strength of the REDOR approach is use of chemically-native proteins and membrane components. The conventional REDOR pulse sequence with 100 kHz 2H π pulses is robust with respect to the 2H quadrupolar anisotropy. The 2H T1’s are comparable to the longer dephasing times (τ’s) and this leads to exponential rather than sigmoidal REDOR buildups. The 13CO-2H buildups are well-fitted to A × (1 − e−γτ) where A and γ are fitting parameters that are correlated as the fraction of molecules (A) with effective 13CO-2H coupling d = 3γ/2. The REDOR approach is applied to probe the membrane locations of the “fusion peptide” regions of the HIV gp41 and influenza virus hemagglutinin proteins which both catalyze joining of the viral and host cell membranes during initial infection of the cell. The HIV fusion peptide forms an intermolecular antiparallel β sheet and the REDOR data support major deeply-inserted and minor shallowly-inserted molecular populations. A significant fraction of the influenza fusion peptide molecules form a tight hairpin with antiparallel N- and C- α helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the β and α fusion peptide structures may be relevant for fusion catalysis via the resultant local perturbation of the membrane bilayer. Future applications of the REDOR approach may include samples that contain cell membrane extracts and use of lower temperatures and dynamic nuclear polarization to reduce data acquisition times.

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“…A similar change in ratio with τ is observed for the monomeric α helical KALP peptide in membranes. 6 …”

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

Jia

Liang

Sackett

et al. 2015

Journal of Magnetic Resonance

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“…The natural abundance ( na ) contributions of lipid DPPC-F16 13 CO nuclei to the displayed S 0 spectra are not considered because these nuclei have shorter transverse relaxation times ( T 2 ) than the HFP 13 CO nuclei. 30 Spin counting yields a ~0.75 fractional contribution of the labeled ( lab ) 13 CO nucleus to the 13 CO S 0 intensity and a ~0.25 na contribution distributed among the 30 unlabeled HFP CO sites. The T 2 of the HFP 13 CO nuclei is ~15 ms with consequent ~0.2 ppm homogeneous contribution to the full-width at half-maximum (FWHM) S 0 13 CO linewidth.…”

Section: Resultsmentioning

confidence: 99%

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

et al. 2013

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Fusion of the HIV membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41 which is an integral membrane protein of HIV. The fusion peptide (FP) is the ~25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of “HFP”, ie. a ~25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was 13CO backbone labeled. Samples were then prepared that each contained a singly 13CO labeled HFP incorporated into membranes which lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP 13CO nuclei and 31P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct 13CO shifts for the α helical and β sheet structures so that the proximities to 31P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the 13CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. “HFPmn” was a linear peptide that contained the 23 N-terminal residues of gp41. “HFPmn_V2E” contained the V2E mutation which for HIV leads to greatly reduced extent of fusion and infection. The present study shows that HFPmn_V2E induces much less vesicle fusion than HFPmn. “HFPtr” contained three strands with HFPmn sequence that were chemically cross-linked near their C-termini. HFPtr mimics the trimeric topology of gp41 and induces much more rapid and extensive vesicle fusion than HFPmn. For HFPmn and HFPtr, well-resolved α and β peaks were observed for A6-, L9-, and L12-labeled samples. For each of these samples, there were similar HFP 13CO to lipid 31P proximities in the α and β structures which evidenced comparable membrane locations of the HFP in either structure including insertion into a single membrane leaflet. The data were also consistent with deeper insertion of HFPtr relative to HFPmn in both the α and β structures. The results supported a strong correlation between the membrane insertion depth of the HFP and its fusogenicity. More generally, the results supported membrane location of the HFP as an important determinant of its fusogenicity. The deep insertion of HFPtr in both the α and β structures provides the most relevant membrane location of the FP for HIV gp41-cat...

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“…In order to probe the membrane location of HFP, the authors measured the distance between the 13 CO of HFP and 31 P of the lipid using REDOR experiments, and revealed a positive correlation between the depth of membrane insertion of HFP and their fusogenicities [78, 189]. A later study enabled the measurement of site-specific membrane locations of HPF by examining the distance between specifically 13 C labeled HFP and specifically 2 H labeled lipids using 13 C- 2 H REDOR experiments [190]. In order to probe the structure of the fusion peptide in large gp41 constructs, Sackett et al built the N70 construct that models a pre-hairpin intermediate, and the FP-Hairpin construct that models the final fusion state with six-helix bundle structure [191, 192].…”

Section: Structure and Dynamics Of Viral Systems With Mas Nmrmentioning

confidence: 99%

Magic angle spinning NMR of viruses

Quinn

Suiter

et al. 2015

Progress in Nuclear Magnetic Resonance Spectroscopy

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Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies.

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Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and 13C–2H rotational-echo double-resonance solid-state NMR

Cited by 12 publications

References 30 publications

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

Magic angle spinning NMR of viruses

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Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and 13C–2H rotational-echo double-resonance solid-state NMR

Cited by 12 publications

References 30 publications

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

Magic angle spinning NMR of viruses

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Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide ¹³CO to Lipid ³¹P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures