Probing Protein Stability with Unnatural Amino Acids

Mendel, David; Ellman, Jonathan A.; Chang, Zhiyuh; Veenstra, David L.; Kollman, Peter A.; Schultz, Peter G.

doi:10.1126/science.1615324

Cited by 145 publications

(84 citation statements)

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“…Both hydrophobicity (Kellis et al, 1988(Kellis et al, , 1989 and packing of interior hydrophobic residues affect protein stability (Sandberg & Terwilliger, 1989;Mendel et al ., 1992). Normally, burying large hydrophobic residues will stabilize the folded structure.…”

Section: Hydrophobic Packing Effects On Protein Stabilitymentioning

confidence: 99%

“…Both hydrophobicity and the packing of hydrophobes in the hydrophobic core of a protein can affect protein stability (Kellis et al, 1988(Kellis et al, , 1989Matsumura et al, 1988;Lim & Sauer, 1989;Sandberg & Terwilliger, 1989;Dill, 1990;Eriksson et al, 1992;Mendel et al, 1992). The role packing effects play in protein folding is still the subject of much debate (Behe et al, 1991).…”

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confidence: 99%

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Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers

et al. 1993

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The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and P-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded a-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HC1. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (AAG values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (AAG values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic a-helices. These structural findings correlate with the observation that in DNAbinding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."Keywords: coiled coils; interchain a-helical interactions; leucine zippers; packing and hydrophobic effects of P-branched amino acids; protein stability It is generally accepted that hydrophobic interactions are the major forces involved in initializing protein folding and stabilizing the three-dimensional structures of proteins. Both hydrophobicity and the packing of hydrophobes in the hydrophobic core of a protein can affect protein stability (Kellis et al

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Section: Hydrophobic Packing Effects On Protein Stabilitymentioning

confidence: 99%

mentioning

confidence: 99%

Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers

et al. 1993

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“…It is not always clear whether adding a larger group to an apparent cavity in a protein will stabilize it, because of the subtle balance between repulsion and dispersion effects (40,41). However, the use of nonnatural amino acids to more precisely fill cavities without experiencing extra van der Waals repulsion should enable one to design more stable proteins (42).…”

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confidence: 99%

What determines the strength of noncovalent association of ligands to proteins in aqueous solution?

Miyamoto

Kollman

1993

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

128

103

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Free energy perturbation methods using molecular dynamics have been used to calculate the absolute free energy of association of two ligand-protein complexes. The calculations reproduce the snificantly more negative free energy of ation of biotin to streptavidin, compared to N-L-acetyltryptophanamlide/a-chymotrypsln. This difference in free energy ofassocition is due to van der Waals/dlspersion effects in the nearly ideally preformed cavity that streptavidin presents to biotin, which involves four ryptophan residues.One of the exciting developments in computer modeling of complex molecules in solution has been the capability to calculate relative free energies of association of these molecules and to relate these values to experiment (1, 2). This development has been catalyzed by methodological advances (3, 4) and increased computer capabilities. In favorable cases, relative free energies of association within 1 kcal/mol (1 cal = 4.184 J) of experiment have been achieved (2). In such cases, the calculations could be of use in experimental ligand design. However, inaccuracies in molecular mechanical force fields and representation of the system and, even more importantly, limitations in one's ability to completely sample the relevant regions of conformational space, have restricted the number of systems to which such free energy calculations could be applied to give chemical accuracy (5,6).Nonetheless, such free energy calculations can be very valuable and interesting even when such accuracy is not achieved, because mechanistic insight into noncovalent association in general and protein-ligand design in particular can be extracted from them (7,8 (14) have had success with this approach for nucleic acid bases in organic solvents, and Lee et al. (15) have calculated the absolute free energy of association of phosphorylcholine analogs to an immunoglobulin, but no one has carried out such a large and dramatic change as in the biotin-streptavidin association studied by Miyamoto and Koilman (9). In that paper, either all of biotin or all of biotin but the terminal COj group were mutated to dummy atoms both in water and in the protein; in either case, a AG for association in the range of -20 kcal/mol could be calculated, in good agreement with experiment. Given the approximations in that study (neglect of any changes in intramolecular energies of biotin free and bound and underestimate of translational/rotational entropy losses due to the use of hydrogen bond restraints) and the difficulty in precisely estimating the magnitude of the errors, we turned to another ligand-protein complex with much lower affinity, with a ligand of size comparable to biotin to use as a control. The results of that calculation, presented here along with the results of the biotin-streptavidin calculation, enable fundamental insights into the nature ofligand-protein associations. METHODSWe used the free energy perturbation method to calculate the binding free energy of complexation. This method uses an easily derived equation from class...

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“…Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.Keywords: 7-azatryptophan; CD studies of staphylococcal nuclease A; 5-hydroxytryptophan; nuclease A (staphylococcal); thermodynamics of unfolding; time-resolved fluorescence studies of staphylococcal nuclease A; tryptophan analogues There has been much interest in recent years in the strategy of incorporating unnatural amino acids into proteins, for example, as a means of specifically perturbing the chemical nature of a particular side chain to test for its contribution to the function of a protein.To cite a few examples, Schultz and coworkers have used a cell-free expression system (chemically attaching the desired unnatural amino acid to suppressor tRNA and then placing the amber codon at the desired position in the mRNA) to incorporate a number of unnatural amino acids, mostly aliphatic amino acid analogues, at various positions in T4 lysozyme (Noren et al, 1989;Ellman et al, 1992;Mendel et al, 1992) and staphylococcal nuclease (Judice et al, 1993;Thorson et al, 1995). The small amount of analogue-containing protein produced was then subjected to…”

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confidence: 99%

“…To cite a few examples, Schultz and coworkers have used a cell-free expression system (chemically attaching the desired unnatural amino acid to suppressor tRNA and then placing the amber codon at the desired position in the mRNA) to incorporate a number of unnatural amino acids, mostly aliphatic amino acid analogues, at various positions in T4 lysozyme (Noren et al, 1989;Ellman et al, 1992;Mendel et al, 1992) and staphylococcal nuclease (Judice et al, 1993;Thorson et al, 1995). The small amount of analogue-containing protein produced was then subjected to…”

mentioning

confidence: 99%

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: Effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

Wong

Eftink

1997

Protein Science

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5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogues of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor. Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate -95% incorporation of 5HW into position 140 of nuclease, and we estimate -98% incorporation of 7AW. CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.Keywords: 7-azatryptophan; CD studies of staphylococcal nuclease A; 5-hydroxytryptophan; nuclease A (staphylococcal); thermodynamics of unfolding; time-resolved fluorescence studies of staphylococcal nuclease A; tryptophan analogues There has been much interest in recent years in the strategy of incorporating unnatural amino acids into proteins, for example, as a means of specifically perturbing the chemical nature of a particular side chain to test for its contribution to the function of a protein.To cite a few examples, Schultz and coworkers have used a cell-free expression system (chemically attaching the desired unnatural amino acid to suppressor tRNA and then placing the amber codon at the desired position in the mRNA) to incorporate a number of unnatural amino acids, mostly aliphatic amino acid analogues, at various positions in T4 lysozyme (Noren et al., 1989;Ellman et al., 1992;Mendel et al., 1992) and staphylococcal nuclease (Judice et al., 1993;Thorson et al., 1995). The small amount of analogue-containing protein produced was then subjected to

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Probing Protein Stability with Unnatural Amino Acids

Cited by 145 publications

References 30 publications

Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers

Packing and hydrophobicity effects on protein folding and stability: Effects of β‐branched amino acids, valine and isoleucine, on the formation and stability of two‐stranded α‐helical coiled coils/leucine zippers

What determines the strength of noncovalent association of ligands to proteins in aqueous solution?

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: Effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

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