Protein Design, a Minimalist Approach

DeGrado, William F.; Wasserman, Zelda R.; Lear, James D.

doi:10.1126/science.2464850

Cited by 489 publications

(292 citation statements)

References 92 publications

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“…Since a number of sets of parameters gave similar x2 values, the possible range of 0 ; s was determined as a range which gives x2 values within 10% of the minimum value. The thermal unfolding profile of disulfide cross-linked tropomyosin was analyzed using the modified local/global unfolding model described above [Eqn (6)]. Since the pretransition was well separated in this case, the parameters AH,", AH,"", tm,, f;: and 0 :…”

Section: Discussionmentioning

confidence: 99%

The local and global unfolding of coiled‐coil tropomyosin

Ishii¹

1994

European Journal of Biochemistry

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The thermal unfolding of a two-stranded a-helical coiled coil of tropomyosin was studied using circular dichroism and excimer fluorescence of N-( 1 -pyrenyl)iodoacetamide-labeled tropomyosin.Tropomyosin unfolds with two transitions, namely local and global unfolding at high salt (greater than 0.1 M NaC1) and pH 7.5. The local unfolding was masked by the global unfolding at low salt (less than 0.1 M NaCl), at high pH (greater than pH 9.0), and in the presence of methanol, where the global unfolding temperature was similar to or lower than the local unfolding temperature. The local and global unfolding are different in nature. A comparison of the helix thermal unfolding of N-( 1 -pyrenyl)iodoacetamide -tropomyosin with unlabeled tropomyosin showed that tropomyosin had an inherent less-stable region, when Cysl90 was N-(1-pyreny1)iodoacetamide-labeled, disulfide cross-linked, or reduced. Instead, the chemical state of Cysl90, determined the stability of the local unfolding region, because a strain created by the disulfide cross-link or the pyrene/pyrene interaction decreases the stability of the local unfolding region. Thus, these data show that the excimer fluorescence of N-(1-pyreny1)iodoacetamide-labeled tropomyosin is useful for studying the local and global unfolding of tropomyosin.a-Helical coiled coils, originally observed in fibrous proteins, are found in many proteins including DNA-binding proteins [l, 21, immunoglobulin-receptor proteins [3], and other proteins. The coiled-coil structure is now recognized as a common stabilizing structural motif for a-helices in proteins [4-61. Tropomyosin is a two-stranded a-helical coiled coil. Differential scanning calorimetry indicates that the tropomyosin molecule consists of cooperative blocks with different thermal stabilities [7, 81. Thermal helix unfolding of tropomyosin exhibits one or two unfolding transitions, depending on the solvent conditions and the chemical state of Cysl90 [9-111. For reduced tropomyosin, a pretransition is indicated by a greater slope in the helix versus temperature profile prior to the main transition with high-salt conditions ; a single transition is observed with low-salt conditions [12]. For disulfide cross-linked tropomyosin, the pretransition and the main transition are clearly distinguished because the disulfide cross-linking decreases the pretransition temperature and increases the main transition temperature [13]. However, it is not clear if the pretransition occurs for reduced tropomyosin as it does for cross-linked tropomyosin. An enzymic digestion method has indicated that the same middle region unfolds for reduced and cross-linked tropomyosin before the main transition, though the transition temperature is different Fluorescence and ESR techniques with probe-labeled tropomyosin have also been used to study the pretransition and the main transition [ 1 2, 15 -171. These techniques provide not only a method to clearly monitor the conformational 1141.Correspondence to Y. Ishii, Biomotron Project, ERATA, Research Development Corp...

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Section: Discussionmentioning

confidence: 99%

The local and global unfolding of coiled‐coil tropomyosin

Ishii¹

1994

European Journal of Biochemistry

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“…However, some tetrameric coiled-coils have been characterized (36,37). X-ray crystallography of GCN4 leucine zipper mutants led Harbury et al (33,34) and others (38) to certain conclusions about the differences in packing in four-stranded coiled-coils compared with the trimeric and dimeric coiled-coils.…”

Section: Two- Three- and Four-stranded Coiled-coil Motifsmentioning

confidence: 99%

α-Helical Protein Assembly Motifs

Kohn

Mant

Hodges

1997

Journal of Biological Chemistry

185

132

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“…Leucine zippers fold into dimeric, parallel coiled-coils, where each heptad forms two a-helical turns. Because of their small size and simple structure, leucine zippers and other short a-helical coiled coils have been used extensively as a model system to study how amino acid sequences specify structure (e.g., see Hodges et al, 1988;DeGrado et al, 1989;Hu et al, 1990;O'Shea et al, 1992O'Shea et al, , 1993Zhou et al, 1992;Pu & Struhl, 1993;Monera et al, 1996;. Leucine zipper-containing transcription factors function as a variety of different pairs of homodimeric and heterodiReprint requests to: James C. Hu, Department of Biochemistry & Biophysics, TexasA&M University, College Station, Texas 77843-2128; e-mail: jimhu@tamu.edu.…”

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Oligomerization properties of GCN4 leucine zipper e and g position mutants

et al. 1997

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Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of >65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing A repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (>dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type I11 mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.Keywords: dimerization specificity; leucine zippers; protein structure; recombinant fusion proteins; site-directed mutagenesis a-Helical coiled coils are involved in the assembly of a wide variety of proteins. A subclass of the coiled-coils known as leucine zippers are found as short dimerization motifs in many eukaryotic transcription factors. Leucine zipper sequences are characterized by leucine appearing in every seventh position (4 over four to five heptad repeats (abcdefg), (for a review, see Hurst, 1994). Leucine zippers fold into dimeric, parallel coiled-coils, where each heptad forms two a-helical turns. Because of their small size and simple structure, leucine zippers and other short a-helical coiled coils have been used extensively as a model system to study how amino acid sequences specify structure (e.g., see Hodges et al., 1988;

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Protein Design, a Minimalist Approach

Cited by 489 publications

References 92 publications

The local and global unfolding of coiled‐coil tropomyosin

The local and global unfolding of coiled‐coil tropomyosin

α-Helical Protein Assembly Motifs

Oligomerization properties of GCN4 leucine zipper e and g position mutants

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