Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

McIntosh, Lawrence P.; Wand, A. Joshua; Lowry, David F.; Redfield, Alfred G.; Dahlquist, Frederick W.

doi:10.1021/bi00479a003

Cited by 88 publications

(88 citation statements)

References 94 publications

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“…Fig. 1 shows part of the NH region of the NOESY spectrum and the corresponding connectivities, while the complete 'H NMR assignment of LYS(l-13) is provided in Table I. As both the crystal and 'H NMR structures of intact T4 lysozyme indicate a well-defined a-helical region spanning residues 3-10 [ [39][40][41], initial efforts were concentrated on detecting signs of the presence of a-helical structures in LYS(l-13). Firstly, a series of weak NH-C&H, together with stronger NH-NH, inter-residue connectivities were seen between residues 3-l 1.…”

Section: Resultsmentioning

confidence: 99%

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

McLeish¹,

Nielsen²,

Wade³

et al. 1993

FEBS Letters

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Solid-phase methods have been used to synthesize LYS(l-13), a peptide corresponding to the first 13 residues of T4 Iysozyme. 2D 'H NMR techniques were used to investigate its solution structure in the presence of SDS micelles. The identification of numerous medium-range NOESY crosspeaks and several slowly exchanging NH protons indicated the presence of an a-helical structure. This was confirmed by simulated annealing calculations performed using XPLOR.

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Section: Resultsmentioning

confidence: 99%

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

McLeish¹,

Nielsen²,

Wade³

et al. 1993

FEBS Letters

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“…Appropriately isotopically labeled cysteine-free T 4 lysozyme (i.e., WT*) and the single-point L99A mutant of WT* were purified as described previously (21,60). Aqueous samples were composed of 500 μM T 4 lysozyme WT* or L99A in 50 mM sodium chloride, 50 mM sodium acetate, pH 5, with 8% (vol/vol) D 2 O.…”

Section: Methodsmentioning

confidence: 99%

Role of cavities and hydration in the pressure unfolding of T₄lysozyme

Nucci

Fuglestad

Athanasoula

et al. 2014

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

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It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T 4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T 4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.protein stability | protein folding and cooperativity | protein hydration | high-pressure NMR | reverse micelle encapsulation

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“…For samples in 95% H20/5% 2H20, the H20 suppression was performed using the SCUBA (20) pulse sequence. Proteins containing amino acids specifically labeled with 15N were used in 1H-15N heteronuclear multiple-quantum coherence experiments (21) to identify the amide protons ofthe labeled amino acids (22). Spectra were obtained using a General Electric GN-500 spectrometer at 500 MHz, except for the Hartmann-Hahn correlated spectra, which were obtained on a Bruker AMX spectrometer at 600 MHz.…”

Section: Methodsmentioning

confidence: 99%

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

Hoffman

Query

Golden

et al. 1991

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

188

100

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An RNA recognition motif (RRM) of '480 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing.The Ul RNA-binding domain ofthe A protein component ofthe human Ul small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel f-strands and two a-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent f-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface of the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins.Over the past decade, the ways in which proteins interact specifically and nonspecifically with DNA have been revealed by biochemical and biophysical studies (reviewed in refs. 1 and 2). Much of this information has been the result of crystallographic analysis, but more recently, NMR techniques have been used (3). As regards RNA-binding proteins, however, there has been little structural information with the notable exception of the work by Steitz and coworkers (4) on the glutaminyl-tRNA synthetase/tRNAGIn complex.Sequence elements characteristic of a group of RNAassociated proteins began to emerge with the observation of four copies of an 80-amino acid repeat in poly(A)-binding protein (5). The most conserved region of 8 amino acids in these repeats was termed the ribonucleoprotein (RNP) consensus sequence, or octamer (RNP1) (6). Another 6-amino acid region was later termed RNP2 (7). It was subsequently shown by several groups that the sequence similarity appears in many other RNA-associated proteins (7-11) (recently reviewed in ref. 12), contains many conserved positions, and is notably rich in aromatic residues (8). Direct evidence of an interaction between this motif and RNA has led to its designation as an RNA recognition motif, or RRM (8). This motif has also been referred to as an RNP consensus sequence-type RNA binding domain (11) and as an RNP-80 motif (13). Members of the RRM-containing family of proteins include heterogeneous nuclear RNP proteins and U-class small nuclear RNP (snRNP) proteins, as well as poly(A)-binding protein, nucleolin, transcription termination factors La and rho, eukaryotic initiation factor 4B, and sexdetermination proteins in Drosophila. The sources of these proteins range from Escherichia coli to man; thus, the RRM is clearly an ancient protein structure.The RRM can be viewed as analogous to the homeobox domain (5) or to the zinc-binding finger motif (14) of DNAbinding proteins. In many cases, the RRM is a modular component ofa larger protein; other m...

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Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

Cited by 88 publications

References 94 publications

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

Role of cavities and hydration in the pressure unfolding of T₄lysozyme

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

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Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

Cited by 88 publications

References 94 publications

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

A peptide corresponding to the N‐terminal 13 residues of T4 lysozyme forms an α‐helix

Role of cavities and hydration in the pressure unfolding of T4lysozyme

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

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Role of cavities and hydration in the pressure unfolding of T₄lysozyme