Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide

Sönnichsen, Frank D.; Eyk, Jennifer E. Van; Hodges, Robert S.; Sykes, Brian D.

doi:10.1021/bi00152a015

Cited by 635 publications

(527 citation statements)

References 55 publications

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“…Interestingly, in the presence of 50% TFE, a helix-inducing solvent (26,31), the K1 peptide reverts to a fully ␣-helical spectrum (equal to that calculated for a 40-residue peptide (23,24). This result indicates that this region of kinesin has the intrinsic ability to adopt either ␤-sheet or helical secondary structures depending on the environment.…”

Section: Analysis Of the Kinesin Neckmentioning

confidence: 77%

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Tripet

Vale

Hodges

1997

Journal of Biological Chemistry

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Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a ϳ50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316 -383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330 -369 displays all of the characteristics of a stable, two-stranded ␣-helical coiled-coil. On the other hand, the NH 2 -terminal segment of the neck (residues ϳ316 -330) has the capacity to adopt a ␤-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370 -383) are not ␣-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended ␣-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiledcoil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.Understanding how motor proteins generate force and movement from the chemical hydrolysis of ATP remains one of the most intriguing problems in biophysics. At present, there are three separate families of motor proteins found within eukaryotic cells: myosins, which move along actin filaments, and kinesins and dyneins, which move along microtubules (1). The motor domains that typify each of these superfamilies exhibit little or no amino acid sequence similarity, and hence it was believed that they had evolved separately and were structurally unrelated. However, the recently determined crystal structure of kinesin revealed an unexpected structural similarity to the core of the myosin motor domain, particularly in the nucleotide binding pocket (2, 3). Hence, myosin and kinesin may share some similarities in how they generate unidirectional movement and force, although the precise mechanistic details remain to be elucidated for both types of motors.Kinesin has proven to be an excellent model system for investigating the mechanism of motility, in part due to the small size of its motor domain (Ͼ2-fold smaller than myosin's). Kinesin purified from tissue sourc...

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Section: Analysis Of the Kinesin Neckmentioning

confidence: 77%

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Tripet

Vale

Hodges

1997

Journal of Biological Chemistry

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“…The effects of alcohols on polypeptides have been found to be strongly sequence-dependent (Lehrman et al, 1990;Segawa et al, 1991;Sonnichsen et al, 1992). The tendency to induce helical structure has been found in nearly all studies, but the amount of alcohol needed varies widely depending on the sequence and the length of the polypeptide.…”

Section: The Mechanism Of Alcohol Denaturationmentioning

confidence: 99%

“…Tamburro et al (1968) studied the effects of trifluoroethanol (TFE) on the conformations of the ribonuclease S-peptide; TFE was found to stabilize the small peptide in the same (a-helical) confor-mation that it adopts in the native protein. Since then, alcohols have been used widely to examine the conformational (particularly helical) propensities of peptides (Nelson & Kallenbach, 1986;Lehrman et al, 1990;Segawa et al, 1991;Sonnichsen et al, 1992) and to induce conformational changes in intact proteins (Stone et al, 1985;Wilkinson & Mayer, 1986;Dufour & HaertlC, 1990;Jackson & Mantsch, 1992;Buck et al, 1993;Fan et al, 1993). The primary physical mechanisms by which alcohols effect these changes are still unresolved.…”

mentioning

confidence: 99%

Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation

1993

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How important are helical propensities in determining the conformations of globular proteins? Using the twodimensional lattice model and two monomer types, H (hydrophobic) and P (polar), we explore both nonlocal interactions, through an HH contact energy, E, as developed in earlier work, and local interactions, through a helix energy, u. By computer enumeration, the partition functions for short chains are obtained without approximation for the full range of both types of energy. When nonlocal interactions dominate, some sequences undergo coil-globule collapse to a unique native structure. When local interactions dominate, all sequences undergo helix-coil transitions. For two different conformational properties, the closest correspondence between the lattice model and proteins in the Protein Data Bank is obtained if the model local interactions are made small compared to the HH contact interaction, suggesting that helical propensities may be only weak determinants of globular protein structures in water. For some HP sequences, varying U/E leads to additional sharp transitions (sometimes several) and to "conformational switching" between unique conformations. This behavior resembles the transitions of globular proteins in water to helical states in alcohols. In particular, comparison with experiments shows that whereas urea as a denaturant is best modeled as weakening both local and nonlocal interactions, trifluoroethanol is best modeled as mainly weakening HH interactions and slightly enhancing local helical interactions.Keywords: alcohol denaturation; compact conformations; helical propensities; HP lattice model; hydrophobic interaction What is the relative importance of local interactions (helical propensities) compared to nonlocal interactions (mainly solvent-mediated and hydrophobic interactions) in determining the native structures of globular proteins? One view has held that the local interactions are the main determinants of structure, while the nonlocal interactions just provide nonspecific stability to the compact state (Anfinsen & Scheraga, 1975). That is, interactions among adjacent and near-neighbor peptide units tend to drive proteins to configure into helices at certain points in the sequence, which ultimately end up as helices in the native structure. In this view, helices should be identifiable by factors that are local within the sequence, and this should strongly specify how they can pack to form the native structure. Recently, an alternative view has developed that nonlocal interactions may be a major determinant not only of the stabilities but also of the structures of globular Reprint requests to: Ken A. Dill, Department of Pharmaceutical Chemistry, Box 1204, University of California, San Francisco, California 94143-1204. proteins Chan & Dill, 1991b). In this view, a major factor in determining where helices form in native structures is the sequence positions of hydrophobic monomers. That is, predicting helices (and also sheets) in native structures is more a matter of finding the...

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“…Fluoroalcohols have been shown to exhibit remarkable smcture stabilizing effects for peptides in aqueous solution (Nelson & Kallenbach, 1989;Bruch et al, 1992;Dyson et al, 1992;Sonnichsen et al, 1992). Enhancements of secondary structure content upon addition of fluoroalcohols have also been demonstrated in a large number of proteins (Alexandrescu et al, 1994;Buck et al, 1995;Shiraki et al, 1995).…”

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confidence: 99%

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

Bhattacharjya

Balaram

1997

Protein Science

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A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced a f f i n i t y towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 'H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (250%). This transition is characterized by a dramatic loss of A N S binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. HID exchange experiments yield higher protection factors for many of the backbone amide protons from the four a-helices along with the C-terminal 3'0 helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel @-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for a-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

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Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide

Cited by 635 publications

References 55 publications

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation

Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

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