ABSTRACTyII-crystailin from calf eye lens consists of two homologous domains, each composed of two similar "Greek key" motifs. As a consequence of the bilobal structure, a biphasic transition is seen upon unfolding by urea at low pH (monitored by circular dichroism, fluorescence emission, and ultracentrifugal analysis). In 3.3 ± 0.5 M urea, a stable intermediate is formed at equilibrium, whereas 5.5 M urea causes maximum denaturation. Unfolding/folding kinetics display a complex pattern characterized by two kinetic phases. Both reactions exhibit strong dependence on the urea concentration; in the range ofthe respective transition, their rates are extremely slow (k = 1 x 10-4 s-'). In conclusion, folding of yHl-crystallin proceeds through the independent sequential structuring of the domains.Eye lens proteins are not subject to the general protein turnover; they remain in their native conformation during the whole life-span of the organism. The molecular mechanism underlying the extreme long-term stability is presently unknown. y-crystallins, the main components ofthe core region ofthe eye lens, have been characterized in detail: the amino acid sequences of most proteins from calf eye lens are known, and for yII-crystallin, the crystal structure has been solved at high resolution (1). The protein is a monomer of 20 kDa that does not contain disulfide bonds; it consists of two homologous domains, each composed of two "Greek key" motifs forming a sandwich of two four-stranded antiparallel a-pleated sheets (2, 3). Because 'yII-crystallin is almost entirely composed of ,l-sheets, this protein should be a good model for the analysis of P-structure formation.'yII-crystallin is a highly stable protein that preserves its native state at pH 1-10. In 0.1 M phosphate buffer, pH 7, the protein is stable to 75°C; at higher temperature, irreversible aggregation occurs. In 7 M urea, denaturation is only seen at elevated temperature or low pH; under these conditions, a two-step equilibrium transition suggests independent domain folding (4).We report here equilibrium and kinetic studies on the domain folding of yII-crystallin. Sedimentation analysis, intrinsic fluorescence, circular dichroism, and thermal analysis prove that the overall reaction at pH 2 can be quantitatively described by a three-state model: MATERIALS AND METHODSPreparation of yII-Crystallin and Its NH2-Terminal Domain. yII-crystallin from calf eye lenses was prepared according to the method of Bjork (5), modified by van Dam (6) and Nesslaier (7). Purity of the protein was ascertained by SDS/PAGE, isoelectric focusing, and spectroscopic techniques: Phast-system (Pharmacia), fluorescence spectrophotometer MPF 44A (Hitachi/Perkin-Elmer), spectropolarimeter J-500/DP-500N (Jasco, Easton, MD). Fast protein liquid chromatographic separations (Pharmacia) made use of the gradient programmer GP-250 with UV-1 monitor.To isolate the NH2-terminal domain fragment, partially unfolded yII-crystallin (obtained by 24-hr incubation of the protein in 3 M urea/0.1 M NaCl/HCl, pH 2....

yII-crystallin from calf eye lens consists of two homologous domains, connected by a six-residue linker peptide. In order to study the intrinsic properties of the domains and their mutual stabilization, limited proteolysis was applied. Optimum conditions providing a homogeneous 10-kDa fragment at high yield were pepsin cleavage in 0.1 M NaCl/HCl pH 2.0, in the presence of 3.0 M urea. Determination of the N-terminus and the C-terminal sequence showed that cleavage occurred at the Phe88 -Arg89 peptide bond, giving rise to the complete N-terminal domain including the connecting hexapeptide. The C-terminal part of the polypeptide chain is cleaved to small fragments.Comparing the spectral properties of the isolated N-terminal domain and intact yII-crystallin proved the structure of the fragment to be closely similar to that of the native domain. Small differences in absorbance, fluorescence emission and circular dichroism point to alterations caused by the increase in surface area as a consequence of domain separation. The resistance of the 1 0-kDa fragment toward thermal and alkaline denaturation, as well as unfolding in the presence of urea or guanidine . HCl is decreased, due to the lack of domain interactions stabilizing the intact protein.Unfolding/folding kinetics of the 10-kDa fragment coincide with the second phase of the bimodal transition of intact yII-crystallin, in agreement with independent sequential folding and modular assembly of the domains within the native molecule. Domains, as distinct structural regions within globular proteins, represent autonomous cooperative folding units. As taken from their occurrence in all large proteins, 'modular assembly' [l] is a general strategy of protein folding offering long polypeptide chains the advantage of rapid self-organization. The discovery of coding and non-coding sequences in genomic DNA, and the correlation of individual protein domains with separate exons suggests 'genes-in-pieces' to imply 'proteins-in-pieces' as a common feature in the processes of transcription, splicing and translation. In certain cases, domains have been shown to originate from gene duplications. In cases where active sites reside on different domains or at domain interfaces, domain interactions may have functional significance, e.g. in terms of hinge movements, or mutual stabilization.yII-Crystallin is a typcial two-domain protein.As taken from high-resolution X-ray analysis, the N-and C-terminal halves of the molecule show twofold symmetry, each of the domains representing a sandwich consisting of two fourstrand pleated sheets organized in 'Greek key' motifs [2]. Both sequence homology and molecular topology suggest that 711-crystallin is the product of gene duplication. The high symmetry and the distinct charge pattern on the surface of the molecule may be responsible for the anomalously high stability of the molecule [3]. Whether, in this context, the domains are intrinsically stable, or whether domain interactions conCorrespondence to R. Jaenicke,

Thermal and GdmC1-induced unfolding transitions of aldolase from Stuphylococcus uureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a AGO value of -9 f 2 kJ/mol at 20 "C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 "C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of a//3 barrel proteins.Keywords: a//3 barrel; folding intermediates; prolyl isomerization; protein folding kinetics; two-state model The mechanism of protein folding has been the subject of intensive studies over the last years. Experimental approaches to the folding problem have mainly focused on the elucidation of folding pathways, the characterization of folding intermediates, and the investigation of the dominant forces in protein stability (for reviews see Jaenicke, 1987;Privalov & Gill, 1988;Kuwajima, 1989;Kim & Baldwin, 1990). It is now widely accepted that protein folding advances through a number of definite intermediate stages (Baldwin, 1990). In the millisecond time region, secondary structural elements and parts of the tertiary contacts are formed (Roder et al., 1988;Udgaonkar & Baldwin, 1988Kuwajima, 1989). Subsequent slow steps are often limited by cis trans isomerization reac- Abbreviations; Aldolase, fructose-l,6-bisphosphate aldolase (EC 4.1.2.13) from Stuphylococcus aureus; GdmC1, guanidinium chloride; N, native state of the protein; U, unfolded protein; UF and Us, fast and slow refolding molecules, respectively; AG, free energy of folding; a, reduced amplitude; X, apparent rate constant; 7, relaxation time.

Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.