NMR Studies of the Structure and Dynamics of Membrane-Bound Bacteriophage Pf1 coat protein

Shon, Ki-Joon; Kim, Yongae; Colnago, Luiz Alberto; Opella, Stanley J.

doi:10.1126/science.1925542

Science

1991

DOI: 10.1126/science.1925542

|View full text |Cite

NMR Studies of the Structure and Dynamics of Membrane-Bound Bacteriophage Pf1 coat protein

Ki-Joon Shon

Yongae Kim

Luiz Alberto Colnago

et al.

Abstract: Filamentous bacteriophage coat protein undergoes a remarkable structural transition during the viral assembly process as it is transferred from the membrane environment of the cell, where it spans the phospholipid bilayer, to the newly extruded virus particles. Nuclear magnetic resonance (NMR) studies show the membrane-bound form of the 46-residue Pf1 coat protein to be surprisingly complex with five distinct regions. The secondary structure consists of a long hydrophobic helix (residues 19 to 42) that spans t… Show more

Help me understand this report

Search citation statements

Order By: Relevance

Paper Sections

Select...

Citation Types

Supporting

Mentioning

130

Contrasting

Year Published

1995

2012

Publication Types

Select...

Article10

Relationship

Self Cite0

Independent10

Authors

Journals

Cited by 170 publications

(140 citation statements)

References 19 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…The approach presented here provides a new approach that shows that the C-and N-terminal parts of these coat proteins contain a large amount of secondary structure that resembles a discontinuous a-helix, indicating that these parts probably arising from an increased amount of molecular motion. This concept is in agreement with conclusions arising from NMR (Shon et al, 1991;Henry & Sykes, 1992; Van de Ven et al, 1993) and FT-IR studies (Thiaudiere et al, 1993).…”

supporting

confidence: 91%

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems

Wolkers¹,

Haris²,

Pistorius³

et al. 1995

Biochemistry

View full text Add to dashboard Cite

FT-IR spectroscopy has been applied to study the secondary structure of the major coat protein of Pfl and M l 3 as present in the phage and reconstituted in DOPG and mixed DOPC/DOPG (4/1) bilayers. Infrared absorbance spectra of the samples were examined in dehydrated films and in suspensions of DiO and H2O. The secondary structure of the coat protein is investigated by second-derivative analysis, Fourier self-deconvolution, and curve fitting of the infrared bands in the amide I region (1600-1700 cm "1), is found that, in dehydrated films o f P fl and M13 phage, the amide I region contains three bands located at about 1633, 1657, and 1683 c m "1, that are assigned to hydrogen-bonded turn, a-helix/random coil, and non-hydrogen-bonded turn, respectively. From a comparison of the infrared spectra in dehydrated film with those in aqueous suspension, the percentages of secondary structure were found with an accuracy of about ±5%. For the coat protein of P fl phage, the FT-IR quantification gives 69% a-helix conformation, 19% turn structure, and 12% random coil structure. For P fl coat protein in the membrane-embedded state, the amount of a-helix is 57%, whereas 42% is in a turn structure and 1% in a random coil structure.

show abstract

supporting

confidence: 91%

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems

Wolkers¹,

Haris²,

Pistorius³

et al. 1995

Biochemistry

View full text Add to dashboard Cite

show abstract

“…was observed (9,11,12 Solid-state NMR is well suited for characterizing proteins in anisotropic environments, and moreover, it can take advantage of uniaxially aligned samples (13)(14)(15)(16)(17)(18). The gramicidin channel structure has been solved with solid-state NMR-derived orientational constraints from samples in fully hydrated lipid bilayers (13).…”

mentioning

confidence: 99%

Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Arumugam

Pascal

North

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The polypeptide gramicidin A is particularly appropriate for these studies of conformational stability, because it has a large surface to volume ratio. Its molecular structure and dynamics are highly sensitive to its environment. In lipid bilayers, gramicidin typically forms a single-stranded helical dimer (see Fig. 1, blue), whereas in organic solvents, it forms a variety of stable double-helical conformations (see Fig. 1, red) that vary in handedness and symmetry (i.e., parallel vs. antiparallel) (7). These conformers interconvert slowly enough in ethanol that four different conformations are well resolved by solution NMR spectroscopy (8). They convert much more slowly in dioxane or tetrahydrofuran, because the solvent cannot efficiently promote hydrogen bond exchange, and consequently, the conformers are trapped in a metastable state (7, 9, 10). In fact, they can be separated by TLC or normal-phase HPLC, and solution NMR spectroscopy can be performed on the individual conformers (10). The equilibrium between these conformational states varies, depending primarily on the dielectric constant of the solvent (unpublished results). Furthermore, when lipid bilayers containing gramicidin were prepared by first cosolubilizing peptide and lipid in various organic solvents followed by solvent removal and hydration, a solvent history dependenceThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.was observed (9,11,12 Solid-state NMR is well suited for characterizing proteins in anisotropic environments, and moreover, it can take advantage of uniaxially aligned samples (13-18). The gramicidin channel structure has been solved with solid-state NMR-derived orientational constraints from samples in fully hydrated lipid bilayers (13). Through isotope labeling of individual specific amino acid sites, orientation-dependent nuclear spin interactions have been monitored in aligned samples (see Fig. 1). Because of the many precise constraints observed for the gramicidin channel, its structure is one of the highest resolution characterizations of a membranebound polypeptide or protein structure. MATERIALS AND METHODSIsotopically labeled amino acids were purchased from Cambridge Isotope Laboratories (Cambridge, MA). Dimyristoyl phosphatidylcholine was purchased from Sigma. Both amino acids and lipids were used without further purification. Gramicidin A was synthesized using fluorenylmethoxycarbonyl blocking chemistry and a peptide synthesizer (Applied Biosystems; model 430A) (19). Peptides that were synthesized in <98% purity were purified by semipreparative HPLC as described (19). Oriented samples were prepared using a gramicidin/lipid molar ratio of 1:8. The peptide and lipid were cosolubilized in an organic solvent, and the sample was dried on thin glass plates. Approximately 20 plates were stacked in a segment of square glass tubing that was sealed following the addition of ...

show abstract

“…Of these methods, deuterium NMR is unique in that it allows direct observation and detailed analysis of internal motions of amino acid side chains both in the solid state (Oldfield & Rothgeb, 1980;Kinsey et al 1981aKinsey et al , 1981bRice et al, 1981;Batchelder et al, 1982;Gall et al, 1982;Keniry et al, 1983Keniry et al, , 1984Baianu et al, 1984;Colnago et al, 1987;Leo et al, 1987;Davis, 1988;Shon et al, 1991) and in solution (Wooten & Cohen, 1979;Akasaka et al, 1988), provided that site-specific deuterium labeling is made. For example, in cytochrome c (Spooner & Watts, 1991), and in filamentous bacteriophage P f l coat protein (Nambudripad et al, 1991), amino acid residues with distinct internal motions have been identified with deuterium NMR in the solid state.…”

mentioning

confidence: 99%

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state

et al. 1996

View full text Add to dashboard Cite

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme-recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core.The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild-type SSI was deuterium-labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8-1.0 x 10"'s indicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 x s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 X lo-* s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side-chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein-water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein-water), became extremely high in amplitude and frequency (> 10 MHz). In contrast, the polycrystalline wild-type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side-chain motions are similar in the fully hydrated powder and in crystals. Spin-lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between IO-' and lO"'s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large-amplitude motions are absent, but the side chains of methionines 7 0 and/or 73 are likely to have small-amplitude motions.Keywords: deuterium NMR; methionyl side chain dynamics; hydration of solid proteins; protein internal motion; specific deuteration; Streptomyces subtilisin inhibitor

show abstract

scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.

Contact Info

customersupport@researchsolutions.com

10624 S. Eastern Ave., Ste. A-614

Henderson, NV 89052, USA

This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.

Blog Terms and Conditions API Terms Privacy Policy Contact Cookie Preferences Do Not Sell or Share My Personal Information

Made with 💙 for researchers

Part of the Research Solutions Family.

NMR Studies of the Structure and Dynamics of Membrane-Bound Bacteriophage Pf1 coat protein

Cited by 170 publications

References 19 publications

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems

Conformational trapping in a membrane environment: a regulatory mechanism for protein activity?

Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state

Contact Info

Product

Resources

About