Experimental investigation of sidechain interactions in early folding intermediates

Engelhard, Markus; Evans, Philip A.

doi:10.1016/s1359-0278(96)00016-8

Cited by 16 publications

(6 citation statements)

References 32 publications

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“…In their present form, all of these probes determine structure incompletely. Nonetheless, the different approaches yield information that is generally complementary, not only mutually but with other slower techniques such as dynamic NMR or pulsed deuterium exchange (42). We first discuss the spectroscopic probes of protein structure and then discuss techniques for fast initiation of folding reactivity.…”

Section: Technical Developmentsmentioning

confidence: 99%

FAST EVENTS IN PROTEIN FOLDING: The Time Evolution of Primary Processes

Callender

Dyer

Gilmanshin

et al. 1998

Annu. Rev. Phys. Chem.

197

170

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Most experimental studies on the dynamics of protein folding have been confined to timescales of 1 ms and longer. Yet it is obvious that many phenomena that are obligatory elements of the folding process occur on much faster timescales. For example, it is also now clear that the formation of secondary and tertiary structures can occur on nanosecond and microsecond times, respectively. Although fast events are essential to, and sometimes dominate, the overall folding process, with a few exceptions their experimental study has become possible only recently with the development of appropriate techniques. This review discusses new approaches that are capable of initiating and monitoring the fast events in protein folding with temporal resolution down to picoseconds. The first important results from those techniques, which have been obtained for the folding of some globular proteins and polypeptide models, are also discussed.

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Section: Technical Developmentsmentioning

confidence: 99%

FAST EVENTS IN PROTEIN FOLDING: The Time Evolution of Primary Processes

Callender

Dyer

Gilmanshin

et al. 1998

Annu. Rev. Phys. Chem.

197

170

View full text Add to dashboard Cite

show abstract

“…To paraphrase Fersht and colleagues, the early events in protein folding currently make up the most obscure and controversial area of folding because these events have been inaccessible to experiment (Prat Gay et al, 1994; also see Evans & Radford, 1994;Engelhard & Evans, 1996), although experiments are beginning to access these time scales (Plaxco & Dobson, 1996;Wolynes et al, 1996;Eaton et al, 1997 and references therein). Computational techniques may also provide information about these experimentallyinaccessible events between the unfolded states and late folding intermediates.…”

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confidence: 99%

Molecular dynamics simulations of hydrophobic collapse of ubiquitin

Alonso

Daggett

1998

Protein Science

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Nine nonnative conformations of ubiquitin, generated during two different thermal denaturation trajectories, were simulated under nearly native conditions (62 "C). The simulations included all protein and solvent atoms explicitly, and simulation times ranged from 1-2.4 ns. The starting structures had a-carbon root-mean-square deviations (RMSDs) from the crystal structure of 4-1 2 8, and radii of gyration as high as 1.3 times that of the native state. In all but one case, the protein collapsed when the temperature was lowered and sampled conformations as compact as those reached in a control simulation beginning from the crystal structure. In contrast, the protein did not collapse when simulated in a 60% methanol: water mixture. The behavior of the protein depended on the starting structure: during simulation of the most native-like starting structures ( 3 8, RMSD to the crystal structure) the RMSD decreased, the number of native hydrogen bonds increased, and the secondary and tertiary structure increased. Intermediate starting structures (5-10 8, RMSD) collapsed to the radius of gyration of the control simulation, hydrophobic residues were preferentially buried, and the protein acquired some native contacts. However, the protein did not refold. The least native starting structures (10-12 8, RMSD) did not collapse as completely as the more native-like structures; instead, they experienced large fluctuations in radius of gyration and went through cycles of expansion and collapse, with improved burial of hydrophobic residues in successive collapsed states.

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“…2A and Table 1). We assume that these early intermediates are comparable to compact folding intermediates that are defined by considerable secondary structure, but are devoid of defined tertiary structure (Fink, 1995 ;Engelhard and Evans, 1996) [compact folding intermediates must not be confused with the Compact conformation (Dornmair et al, 1989) of native class I1 MHC molecules]. Compact folding intermediates may thus be formed in the pres-ence of mild detergents, or in the presence of low, but not high, concentrations of aggressive detergents such as SDS.…”

Section: Discussionmentioning

confidence: 99%

Pathway of Detergent‐Mediated and Peptide Ligand‐Mediated Refolding of Heterodimeric Class II Major Histocompatibility Complex (MHC) Molecules

Stöckel

Döring

Malotka

et al. 1997

European Journal of Biochemistry

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We investigated the mechanism of refolding and reassembly of recombinant a and p chains of the class 11 major histocompatibility molecules (MHC-11) HLA-DRB5*0101. Both chains were expressed in the cytosol of Esckerichin coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the a and p chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C, to C,, in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than shortchain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.Keywords: major histocompatibility complex; protein folding ; folding intermediate; heterodimer formation; detergent.In vitro and in living cells most proteins fold by passing a series of intermediates where at each stage an increase of structural organization is gained (Jaenicke, 1991 ;Matthews, 1993;Rudolph and Lilie, 1996). During a folding reaction hydrophobic regions are transiently exposed to water. This makes the intermediates susceptible to aggregation which is the dominant competing reaction of protein folding. In living cells aggregation is prevented by chaperone molecules (Ellis and Hartl, 1996). I n vitro, aggregation may be reduced by addition of cosolvents (Sawano et al., 1992;Cleland et al., 1992; Sirokmlin and Fasman, 1993;Buchner and Rudolph, 1991) MHC-IT. class-I1 major histocompatibility complex; HexOGlc, n-hexyl /I-i~-glucoside; OctOGlc, n-octyl 11-D-glucoside; DodOGlc, ii-dodecyl /j-u-glucaside; OctOGlc,, n-octyl /I-D-ghC0-4-(1-glucoside; DodOGlc2, n-dodecyl ~-u-gluco-4-u-glucoside; TetOGlc,, 12-tetradecyl /I-n-gluco-4-a-gluco...

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Experimental investigation of sidechain interactions in early folding intermediates

Cited by 16 publications

References 32 publications

FAST EVENTS IN PROTEIN FOLDING: The Time Evolution of Primary Processes

FAST EVENTS IN PROTEIN FOLDING: The Time Evolution of Primary Processes

Molecular dynamics simulations of hydrophobic collapse of ubiquitin

Pathway of Detergent‐Mediated and Peptide Ligand‐Mediated Refolding of Heterodimeric Class II Major Histocompatibility Complex (MHC) Molecules

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