[3] Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance

Muchmore, David C.; McIntosh, Lawrence P.; Russell, Chris B.; Anderson, David E.; Dahlquist, Frederick W.

doi:10.1016/0076-6879(89)77005-1

Cited by 479 publications

(324 citation statements)

References 64 publications

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“…Notwithstanding the introduction of up to five additional alanines, all of the mutant lysozymes behaved normally and could be purified by the standard procedure (Muchmore et al, 1989;Poteete et al, 1991).…”

Section: Resultsmentioning

confidence: 99%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within a-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the a-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions.All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest ( 5 0 . 4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133 +Ala made a-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino-and carboxyterminal domains changed by 3.6". This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.Keywords: alanine; lysozyme; protein folding; protein structure; thermostability In a systematic attempt to identify residues important in the folding and stability of phage T4 lysozyme we previously substituted four alanines within the a-helix that includes residues 126-134 . The helix is amphipathic, located on the surface of the carboxy-terminal domain, and is remote from the active site ( Fig. 1; Kinemage 1). In wild-type lysozyme this a-helix already includes three alanines. It was found that substitution of three solvent-exposed residues, Glu 128, Val 131, and Asn

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Section: Resultsmentioning

confidence: 99%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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“…All experiments used the catalytic domain of Cex and its mutants, expressed and purified according to methods°adapted°from°earlier°procedures° [21][22][23].°Details°are given in the Appendix. The final stock solution concentrations of all the proteins were about 1-4 mg/mL.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…The plasmids were transformed into E. coli BL21( DE3) cells and cultured on agar media containing ampicillin with the resultant colonies selected for growth in liquid LB media and stored at Ϫ80°C. Wild-type Cex and its mutants were identically produced starting with a 20 mL overnight culture in a synthetic°medium° [23]°containing°ampicillin.°A°10°mL sample of the resultant culture was used to inoculate 1 L of synthetic medium, grown to an OD600 of ϳ0.5, induced with 0.1 mM IPTG, and harvested 48 h after induction. The temperature was 30°C throughout the entire growth and expression period.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

Gas phase noncovalent protein complexes that retain solution binding properties: Binding of xylobiose inhibitors to the β-1, 4 exoglucanase from Cellulomonas fimi

Tešić

Wicki

Poon

et al. 2007

J. Am. Soc. Mass Spectrom.

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Tandem mass spectrometry has been used to compare gas-phase and solution binding of three small-molecule inhibitors to the wild type and three mutant forms of the catalytic domain of Cex, an enzyme that hydrolyses xylan and xylo-oligosaccharides. The inhibitors, xylobiosyldeoxynojirimycin, xylobiosyl-isofagomine lactam, and xylobiosyl-isofagomine consist of a common distal xylose linked to different proximal aza-sugars. The three mutant forms of the enzyme contain the substitutions Asn44Ala, Gln87Met, and Gln87Tyr that alter the binding interactions between Cex and the distal sugar of each inhibitor. An electrospray ionization (ESI) triple quadrupole MS/MS system is used to measure the internal energies, ⌬E int , that must be added to gas-phase ions to cause dissociation of the noncovalent enzyme-inhibitor complexes. Collision cross sections of ions of the apo-enzyme and enzyme-inhibitor complexes, which are required for the calculations of ⌬E int , have also been measured. The results show that, in the gas phase, enzyme-inhibitor complexes have more compact, folded conformations than the corresponding apo-enzyme ions. With the mutant enzymes, the effects of substituting a single residue can be detected. The energies required to dissociate the gas-phase complexes follow the same trend as the values of ⌬G 0 for dissociation of the complexes in solution. This trend is observed both with different inhibitors, which probe binding to the proximal sugar, and with mutants of Cex, which probe binding to the distal sugar. Thus the gas-phase complexes appear to retain much of their solution binding characteristics. (J Am Soc Mass Spectrom 2007, 18, 64 -73)

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“…Use of REDOR filtering for larger proteins will require incorporation of specific 13 C and 15 N labels in an expression protocol [2,41,[62][63][64]. A single carbonyl site can be probed using the carboxylic 13 C-labeled amino acid of the residue of interest and the 15 N-labeled amino acid of the next residue in the sequence.…”

Section: Future Applicationsmentioning

confidence: 99%

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Yang

Parkanzky

Bodner

et al. 2002

Journal of Magnetic Resonance

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Clean MAS observation of 13 C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13 C-15 N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13 C carbonyl and its directly bonded 15 N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic-and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13 C, 15 N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13 C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

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[3] Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance

Cited by 479 publications

References 64 publications

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Gas phase noncovalent protein complexes that retain solution binding properties: Binding of xylobiose inhibitors to the β-1, 4 exoglucanase from Cellulomonas fimi

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

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