Similar Hydrophobic Replacements of Leu99 and Phe153 within the Core of T4 Lysozyme Have Different Structural and Thermodynamic Consequences

Eriksson, Anders; Baase, W.A.; Matthews, B.W.

doi:10.1006/jmbi.1993.1077

Cited by 204 publications

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“…The mutants that gave crystals suitable for X-ray analysis were grown under conditions similar to those for wild-type (Le., -2 M phosphate solutions, pH -6.7) (Eriksson et al, 1992(Eriksson et al, , 1993. The locations of these mutations are shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

et al. 1998

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To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts 5 0.3 A); in other cases mainchain atoms moved up to 2 A.In the case of Ile 29 -+ Ala the structure col!apsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 4 Ala (V149A) and Met 6 -+ Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the. cavity. The Val 149 -+ Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 -+ Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu + Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol" k 3 . Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu + Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).

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Section: Resultsmentioning

confidence: 99%

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

et al. 1998

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“…Because a significant proportion of interface surface is constituted by solvated cavities, inclusion of solvent in such simulations is also strongly recommended. The creation of cavities in proteins has been shown to be destabilizing to the free energy of folding (Kellis et al, 1988(Kellis et al, , 1989Karpusas et al, 1989;Eriksson et al, 1992Eriksson et al, , 1993. However, it is difficult to quantify the destabilization directly due to cavity formation and reported values differ (Rashin et al, 1986;Eriksson et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Cavities and packing at protein interfaces

1994

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An analysis of internal packing defects or "cavities" (both empty and water-containing) within protein structures has been undertaken and includes 3 cavity classes: within domains, between domains, and between protein subunits. We confirm several basic features common to all cavity types but also find a number of new characteristics, including those that distinguish the classes. The total cavity volume remains only a small fraction of the total protein volume and yet increases with protein size. Water-filled "cavities" possess a more polar surface and are typically larger. Their constituent waters are necessary to satisfy the local hydrogen bonding potential. Cavitysurrounding atoms are observed to be, on average, less flexible than their environments. Intersubunit and interdomain cavities are on average larger than the intradomain cavities, occupy a larger fraction of their resident surfaces, and are more frequently water-filled. We observe increased cavity volume at domain-domain interfaces involved with shear type domain motions. The significance of interfacial cavities upon subunit and domain shape complementarity and the protein docking problem, as well as in their structural and functional role in oligomeric proteins, will be discussed. The results concerning cavity size, polarity, solvation, general abundance, and residue type constituency should provide useful guidelines for protein modeling and design.

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“…Although substitutions of aromatic residues with Leu are usually tolerated during evolution (Bordo & Argos, 1991), single replacements of this type have been found to destabilize folded proteins significantly (Kellis et al, 1988;Goldenberg et al, 1989;Coplen et al, 1990;Eriksson et al, 1993). The replacement decreases the size of the side chain and eliminates any interaction specific for aromatic rings.…”

Section: Design and Initial Characterization Of Aromatic + Leu Bpti Vmentioning

confidence: 99%

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

Zhang

Goldenberg

1997

Protein Science

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The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.Keywords: aromatic residues; bovine pancreatic trypsin inhibitor; conformational funnels; disulfide bonds; hydrophobic residues; protein foldingThe identification and characterization of partially folded intermediate states have historically been major goals in the study of protein folding mechanisms. Early experiments demonstrating the transient accumulation of such species provided important evidence supporting the view that proteins fold via non-random pathReprint requests to: David P. Goldenberg, Department of Biology, University of Utah, Salt Lake City, UT 84112; e-mail: goldenberg@ bioscience.utah.edu.'Current address: Department of Pathology, Harvard University Medical School, Boston, MA 021 15.Abbreviations: BPI?, bovine pancreatic trypsin inhibitor. Amino acid replacements are indicated by the wild-type residue type (using the oneletter code for the 20 standard amino acids), followed by the residue number and the mutant residue type. The disulfides of native BF'TI and folding intermediates are indicated by the residue numbers of the disulfidebonded cysteine residues; N;;, native-like two-disulfide intermediate containing the 30-51 and 5-55 disulfides. Other intermediates are indicated by square brackets enclosing the disulfide bonds they contain; GSSG, oxidized glutathione; DTT:;, the dithiol form of dithiothreitol; GuHCl, guanidinium chloride; IAEDANS, N-iodoacetyl-N"(5-sulfo-l-naphthyl)ethylenediami...

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Similar Hydrophobic Replacements of Leu99 and Phe153 within the Core of T4 Lysozyme Have Different Structural and Thermodynamic Consequences

Cited by 204 publications

References 0 publications

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

Cavities and packing at protein interfaces

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

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