Incorporation of Isotopically Enriched Amino Acids

Samuel‐Landtiser, Mini; Zachariah, Cherian; Williams, Chris R.; Edison, Arthur S.; Long, Joanna

doi:10.1002/0471140864.ps2603s47

Cited by 5 publications

(5 citation statements)

References 38 publications

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“…For larger molecular systems, the isotopic labeling arrangements required for DQ-DRAWS are amenable to protein expression using Escherichia coli and selecting unique pairs of adjacent amino acids. 63 Even though the work presented here has focused on microcrystalline samples, it should be emphasized that the applicability of the DQ-DRAWS technique can be extended to solid states lacking long-range order. 5,71 The integrity of the experiment is uncompromised for samples in amorphous or glassy states, and may in fact yield important structural information when only local order is present, albeit for unresolved resonances the information would be more qualitative than quantitative.…”

Section: Discussionmentioning

confidence: 99%

“…Isotopically enriched Fmoc amino acids were synthesized starting with 13 C′-enriched amino acids (Cambridge Isotope Laboratories, Inc.) and using established synthesis procedures. 63 After the solid-phase synthesis, peptides were cleaved with 95%:5%::CH2Cl2:TFA and filtered, and the solvent was removed under vacuum. Then crude peptides were brought up in acetic acid and loaded onto Dowex resin, from which, after several water washes, the samples were eluted with 1% ammonium hydroxide and purity was verified by TLC and size exclusion chromatography.…”

Section: Experimental Methodsmentioning

confidence: 99%

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Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

et al. 2008

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Several approaches for utilizing dipolar recoupling solid-state NMR (ssNMR) techniques to determine local structure at high resolution in peptides and proteins have been developed. However, many of these techniques measure only one torsion angle or are accurate for only certain classes of secondary structure. Additionally, the efficiency with which these dipolar recoupling experiments suppress the deleterious effects of chemical shift anisotropy (CSA) at high magnetic field strengths varies. Dipolar recoupling with a windowless sequence (DRAWS) has proven to be an effective pulse sequence for exciting double-quantum (DQ) coherences between adjacent carbonyl carbons along the peptide backbone. By allowing this DQ coherence to evolve, it is possible to measure the relative orientations of the CSA tensors and subsequently use this information to determine the Ramachandran torsion angles phi and psi. Here, we explore the accuracies of the assumptions made in interpreting DQ-DRAWS data and demonstrate their fidelity in measuring torsion angles corresponding to a variety of secondary structures irrespective of hydrogen-bonding patterns. It is shown how a simple choice of isotopic labels and experimental conditions allows accurate measurement of backbone secondary structures without any prior knowledge. This approach is considerably more sensitive for determining structure in helices and has comparable accuracy for beta-sheet and extended conformations relative to other methods. We also illustrate the ability of DQ-DRAWS to distinguish between structures in heterogeneous samples.

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Section: Discussionmentioning

confidence: 99%

Section: Experimental Methodsmentioning

confidence: 99%

Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

et al. 2008

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“…Isotopically enriched Fmoc amino acids were synthesized starting with 13 C′-enriched amino acids (Cambridge Isotope Laboratories, Inc.) and using standard protocols . KL 4 (KLLLLKLLLLKLLLLKLLLLK) was synthesized via automated solid-phase peptide synthesis on a Wang resin (ABI 430, ICBR, University of Florida).…”

Section: Methodsmentioning

confidence: 99%

The Helical Structure of Surfactant Peptide KL₄ When Bound to POPC: POPG Lipid Vesicles

et al. 2008

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KL 4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein B which successfully lowers surface tension in the alveoli. A mechanistic understanding of how KL 4 affects lipid properties has proven elusive as the secondary structure of KL 4 in lipid preparations has not been determined at high resolution. The sequence of KL 4 is based on the C-terminus of SP-B, a naturally occurring helical protein that binds to lipid interfaces. The spacing of the lysine residues in KL 4 precludes the formation of a canonical amphipathic α-helix; qualitative measurements using Raman, CDc and FTIR spectroscopies have given conflicting results as to the secondary structure of the peptide as well as its orientation in the lipid environment. Here, we present a structural model of KL 4 bound to lipid bilayers based on solid state NMR data. Double-quantum correlation experiments employing 13 C-enriched peptides were used to quantitatively determine the backbone torsion angles in KL 4 at several positions. These measurements, coupled with CD experiments, verify the helical nature of KL 4 when bound to lipids, with (Φ, Ψ) angles that differ substantially from common values To whom correspondence should be addressed. Tel: 352-846-1506. Fax:352-392-3422. E-mail: jrlong@mbi.ufl for α-helices of (-60, -45). The average torsion angles found for KL 4 bound to POPC: POPG lipid vesicles are (-105, -30); this deviation from ideal α-helical structure allows KL 4 to form an amphipathic helix at the lipid interface.Keywords pulmonary surfactant; Surfactant Protein B; KL 4 ; sinapultide; lucinactant; peptide secondary structure; alpha-helix; hydrophobic moment; lipid bilayers; membrane proteins; phosphatidylcholine; phosphatidylglycerol; solid state NMR; dipolar recoupling; magic angle spinning Pulmonary surfactant is a lipid-rich fluid, containing key proteins, forming the inner mucosal lining of the alveoli. The primary functions of lung surfactant are to minimize surface tension at the alveolar air-fluid interface and provide a barrier against disease (1-5). Inadequate protein levels are a leading cause of respiratory distress syndrome (RDS) in premature infants (6-8).Current therapies for RDS primarily rely on administration of lung surfactant from exogenous sources (9-12). This reliance on xenogenic surfactant is due to the critical role of lung surfactant protein B (SP-B), a highly hydrophobic, 79 residue protein which functions as a homodimer containing 7 disulfide bridges (13). A multitude of roles for SP-B have been proposed and experimentally established. These include surface tension minimization, facilitation of lipid adsorption and resorption at the air-fluid interface, intracellular surfactant trafficking and improved respiratory dynamics in general (14).Synthetic, peptide-based lung surfactant replacements have received noticeable attention (15-17) as the use of synthetic analogs would remove the immunologic risks associated with animal-derived surfactant and allow for greater therapeutic consistency ...

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“…Selectively deuterated 5- d 3 -L-leucine was purchased (Cambridge Isotopes, Andover, MA) and fmoc-protected using standard protocols [33]. Four variants of KL 4 , each containing a single enriched leucine (at Leu3, Leu10, Leu12, or Leu19), were synthesized via solid-phase peptide synthesis on a Wang resin (ABI 430, ICBR, UF), cleaved from the resin with 90% TFA/5% triisopropyl-silane/5% water and ether precipitated.…”

Section: Methodsmentioning

confidence: 99%

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Long

Mills

Ganesh

et al. 2010

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Summary Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL4 is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL4 is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL4 in phospholipid bilayers, we introduced CD3-enriched leucines at four positions along the peptide to serve as probes of sidechain dynamics via 2H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine sidechains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL4 in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine sidechain dynamics, is consistent with monomeric KL4 lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL4 relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL4. The unusual secondary structure of KL4 and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.

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Incorporation of Isotopically Enriched Amino Acids

Cited by 5 publications

References 38 publications

Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

The Helical Structure of Surfactant Peptide KL₄ When Bound to POPC: POPG Lipid Vesicles

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

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Incorporation of Isotopically Enriched Amino Acids

Cited by 5 publications

References 38 publications

Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

Determination of Peptide Backbone Torsion Angles Using Double-Quantum Dipolar Recoupling Solid-State NMR Spectroscopy

The Helical Structure of Surfactant Peptide KL4 When Bound to POPC: POPG Lipid Vesicles

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

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The Helical Structure of Surfactant Peptide KL₄ When Bound to POPC: POPG Lipid Vesicles