Structural Characterization of a Partly Folded Apomyoglobin Intermediate

Hughson, Frederick M.; Wright, Peter E.; Baldwin, Robert L.

doi:10.1126/science.2218495

Cited by 746 publications

(784 citation statements)

References 36 publications

Supporting

Mentioning

709

Contrasting

Unclassified

Order By: Relevance

“…To support this view experimentally, a comprehensive description on the structure of protein in a full range of the conformational space is necessary. The D, MG, and H conformers and their variants of various proteins have been characterized extensively by many experimental methods (Roder et al, 1988;Hughson et al, 1990;Dill & Shortle, 1991;Jeng & Englander, 1991;Sosnick & Trewhella, 1992;Fan et al, 1993;Alexandrescu et al, 1994;Konno et al, 1995). However, limited sensitivity and resolution of any experimental methods make this problem still largely unsolved.…”

mentioning

confidence: 99%

Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

Konno

1998

Protein Science

View full text Add to dashboard Cite

The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2S04r sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.

show abstract

mentioning

confidence: 99%

Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

Konno

1998

Protein Science

View full text Add to dashboard Cite

show abstract

“…According to recent temperature-jump experiments, secondary structure formation and compaction of the chain in these regions occurs on a sub-millisecond time scale (Ballew et al, 1996). The A, G, and H helices are also folded and packed within a compact molten globule state formed at equilibrium at pH 4 (Hughson & Baldwin, 1989;Hughson et al, 1990). Studies on the conformation of several peptide fragments of myoglobin have already been carried out (Epand & Scheraga, 1968;Hermans & Puett, 1971;Waltho et al, 1989) and an extensive study has been made of the conformational preferences of synthetic peptides of the GH region (Shin et al, 1993a(Shin et al, , 1993bWaltho et al, 1993).…”

mentioning

confidence: 99%

Folding propensities of peptide fragments of myoglobin

et al. 1997

Self Cite

View full text Add to dashboard Cite

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthesized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformational preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the a region of (c#J,$) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A-and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate p-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, earlier studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.Keywords: myoglobin; nuclear magnetic resonance; peptide conformation; protein folding; reverse turn; secondary structureThe mechanism by which proteins fold into their native conformations remains one of the central unsolved problems in molecular biology. One view is that folding occurs along defined pathways, often with structured intermediates (for reviews, see Creighton,

show abstract

“…Sensitivityenhanced 1 H-15 N HSQC experiments (28) were performed at 30°C on a Varian INOVA600 MHz spectrometer equipped with a 1 H{ 13 C, 15 N} triple resonance probe with a triple axis gradient. Each peptide addition was followed by thermal equilibration in the spectrometer for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…Proton chemical shifts were referenced relative to internal 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). 15 N chemical shifts were referenced indirectly as described (31). Resonance assignments are reported elsewhere (27).…”

Section: Methodsmentioning

confidence: 99%

“…Its typical globin fold is extensively represented across all three kingdoms of life (13). Sperm whale apoMb is the best characterized member of the globin family, in terms of its structure, folding mechanisms, and interaction with chaperones (11,(14)(15)(16)(17)(18)(19). This protein is routinely produced in bacterial expression systems (20,21), and it is a suitable model for investigating substrate interactions with chaperones, because portions of its sequence are known to severely self-associate in the absence of chaperones (19).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Binding Specificity of an α-Helical Protein Sequence to a Full-Length Hsp70 Chaperone and Its Minimal Substrate-Binding Domain

et al. 2006

View full text Add to dashboard Cite

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co-and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-R-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substratebinding domain, but relative affinities differ. The binding specificity of DnaK-is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding ∆G°of 3.1 ( 0.9 kcal mol -1 and an m value of 1.7 ( 0.4 kcal mol -1 M -1 .

show abstract

Structural Characterization of a Partly Folded Apomyoglobin Intermediate

Abstract: To understand why proteins adopt particular three-dimensional structures, it is important to elucidate the hierarchy of interactions that stabilize the native state

Cited by 746 publications

References 36 publications

Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

Conformational diversity of acid‐denatured cytochrome c studied by a matrix analysis of far‐UV CD spectra

Folding propensities of peptide fragments of myoglobin

Binding Specificity of an α-Helical Protein Sequence to a Full-Length Hsp70 Chaperone and Its Minimal Substrate-Binding Domain

Contact Info

Product

Resources

About