Photoincorporation of fluorescent probe into GroEL: defining site of interaction.

We have elucidated conditions for the covalent incorporation of a nonspecific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in response to various effectors. This method should be useful for studying both protein structure and dynamics.

Conditions for nucleotide-dependent GroES-GroEL interactions. GroEL14(groES7)2 is favored by an asymmetric distribution of nucleotides.

A still unresolved question regarding the mechanism of chaperonin-assisted protein folding involves the stoichiometry of the GroEL-GroES complex. This is important, because the activities of the Escherichia coli chaperonin GroEL are modulated by the cochaperonin GroES. In this report, the binding of GroES to highly purified GroEL in the presence of ATP, ADP, and the nonhydrolyzable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (AMP-PNP), was investigated by using the fluorescence anisotropy of succinimidyl-1-pyrenebutyrate-labeled GroES. In the presence of Mg2+-ATP and high [KCl] (10 mM), two GroES7 rings bind per one GroEL14. In contrast, in the presence of ADP or AMP-PNP only one molecule of oligomeric GroES can be tightly bound by GroEL. With AMP-PNP, binding of a small amount (<20%) of a second GroES can be detected. In the presence of ADP alone, a second GroES ring can bind to GroEL weakly and with negative cooperativity. Strikingly, addition of AMP-PNP to the solution containing preformed GroEL14(GroES7) complexes formed in the presence of ADP results in an increase in the fluorescence anisotropy. Analysis of this effect indicates that 2 mol of GroES oligomer can be bound in the presence of mixed nucleotides. A similar conclusion follows from studies in which ADP is added to an GroEL14 (GroES7) complex formed in the presence of AMP-PNP. This is the first demonstration of an asymmetric distribution of nucleotides bound on the 1:2 GroEL14 (GroES7)2 complex. The relation of the observed phenomena to the proposed mechanism of the GroEL function is discussed.

Preformed GroES oligomers are not required as functional cochaperonins.

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268-30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96-97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer-monomer equilibrium to be 7.3 x 10(-34)M6. The GroESD(96-97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96-97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the performed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.

Reversible oligomerization and denaturation of the chaperonin GroES.

The chaperonin GroEL can assist protein folding and normally acts with the co-chaperonin GroES. These Escherichia coli proteins are encoded on the same operon, with GroES positioned first. In this report, we have investigated the reversible folding of GroES. Using fluorescence anisotropy of dansyl-labeled GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity, and limited proteolysis, we show that GroES unfolds in a single, two-state transition. Importantly, intrinsic fluorescence and sedimentation velocity analyses show that GroES is capable of refolding and reassembling from a urea denatured state. The refolded GroES is fully active as shown by its ability to assist GroEL in the refolding of rhodanese. These results indicate that chaperonins may not require other chaperonins for successful folding/assembly. We also show that GroES is capable of assisting in the refolding/reassembly of fully denatured GroEL. The reversible folding of GroES coupled with the ability of GroES to assist the refolding/reassembly of GroEL suggest that the groE operon may be organized in a manner that provides a structural role in GroES/GroEL assembly as well as a functional role.

The C-terminal sequence of the chaperonin GroES is required for oligomerization.

The Escherichia coli protein GroES is a co-chaperonin that is able to assist GroEL in the refolding of proteins. GroES is a heptamer of seven identical subunits. Recent work has focused on the structural aspects of GroES. We have investigated the role of the C-terminal portion of GroES on its oligomerization. Limited proteolysis of GroES by carboxypeptidase Y gives a product in which the C-terminal 7 amino acid residues have been removed. Sedimentation velocity analysis reveals that the truncated form of GroES is unable to reassemble. The results presented here implicate the C-terminal sequence in intermonomer actions within the GroES oligomer. In addition, this work provides experimental verification of predictions implied in the recent x-ray determination of the GroES structure (Hunt, J. F., Weaver, A. J., Landry, S. J., Gierasch, L. M., and Deisenhofer, J. Nature, in press).

Residual structure in urea-denatured chaperonin GroEL.

The urea denaturation of the chaperonin GroEL has been studied by circular dichroism, intrinsic tyrosine fluorescence and fluorescence of the hydrophobic probe, 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). It is shown that GroEL denaturation, monitored by CD and intrinsic fluorescence measurements, can be well described by a two-state transition that is complete by 3-3.1 M urea. The beginning of this transition overlaps the urea concentrations where the oligomeric protein starts to dissociate into individual monomers. Subsequent addition of the denaturant leads to complete unfolding of the monomers. Monomers unfolded at urea concentrations higher than 3.1 M are not competent to form their native conformations under the conditions employed here, and they are not able to reassemble to oligomers upon dilution of urea. In contrast to the CD and intrinsic fluorescence measurements, bisANS bound to GroEL exhibits considerable fluorescence intensity under conditions where the CD and intrinsic fluorescence signals have already reached their minimum values (> 3.1 M urea). This binding of bisANS, under conditions where the majority of the secondary structure of GroEL has already unfolded, indicates the existence of hydrophobic residual structure. This structure cannot be detected by CD measurements, but it can be unfolded by raising further the urea concentration. The existence of this structure does not depend on the source or method of the protein preparation. Intrinsic fluorescence and trypsin digestion demonstrate no difference between the bisANS-bound form of GroEL and the free form of the protein, showing that the GroEL structure is not greatly affected by the interaction with bisANS.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoincorporation of 4,4'-bis(1-anilino-8-naphthalenesulfonic acid) into the apical domain of GroEL: specific information from a nonspecific probe.

The use of noncovalent hydrophobic probes such as bis-ANS has become increasingly popular in gaining structural information about protein structure and conformation. While these probes have provided rich information about protein conformation, specific information has been limited. In this report, we extend the usefulness of the probe bis-ANS by showing that it can be covalently photoincorporated into various proteins. Using the chaperonin GroEL, we have shown that it is possible to locate important hydrophobic surfaces through photoincorporation and peptide sequencing. It has been proposed that hydrophobic surfaces on the chaperonin may be responsible for the binding of unfolded polypeptides. We show here that photoincorporation of bis-ANS is able to locate a distinct hydrophobic surface on GroEL. Incorporation of the bis-ANS occurs within a 45 residue fragment of the monomer near the middle of the primary sequence. Interestingly, photoincorporation occurs within this fragment in both tetradecamers and assembly-competent monomers. From the three-dimensional structure of GroEL, this region maps to the apical domain (residues 191-376), which has been implicated in polypeptide binding [Fenton, W. A., Kashi, Y., Furtak, K., & Horwich, A. L. (1994) Nature 371, 614-619]. In addition, the fluorescent properties of the probe are retained including the excitation and emission maxima and the sensitivity to the polarity of its environment. These results suggest that photoincorporated bis-ANS may be a useful probe for protein structure and dynamics.

Sequence determinants of the capping box, a stabilizing motif at the N-termini of alpha-helices.

The capping box, a recurrent hydrogen bonded motif at the N-termini of alpha-helices, caps 2 of the initial 4 backbone amide hydrogen donors of the helix (Harper ET, Rose GD, 1993, Biochemistry 32:7605-7609). In detail, the side chain of the first helical residue forms a hydrogen bond with the backbone of the fourth helical residue and, reciprocally, the side chain of the fourth residue forms a hydrogen bond with the backbone of the first residue. We now enlarge the earlier definition of this motif to include an accompanying hydrophobic interaction between residues that bracket the capping box sequence on either side. The expanded box motif--in which 2 hydrogen bonds and a hydrophobic interaction are localized within 6 consecutive residues--resembles a glycine-based capping motif found at helix C-termini (Aurora R, Srinivasan R, Rose GD, 1994, Science 264:1126-1130).