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Proteins can adopt totally different folded conformations1

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Abstract

The three-dimensional structure of a protein is determined by interactions between its amino acids and by interactions of the amino acids with molecules of the environment. The great influence of the latter interactions is demonstrated for the enzyme phosphoglycerate kinase from yeast (PGK). In the native state, PGK is a compact, bilobal molecule; 35 % and 13 % of its amino acids are organised in the form of α-helices and β-sheets, respectively. The molecules unfold at acidic pH and low ionic strength forming random-walk structures with a persistence length of 3 nm. More than 90 % of the amino acid residues of the ensemble have φ,ψ-angles corresponding to those of a straight β-chain. Upon addition of 50 % (v/v) trifluoroethanol to the acid-unfolded protein, the entire molecule is transformed into a rod-like, flexible α-helix. Addition of anions, such as chloride or trichloroacetate, to the acid-unfolded protein leads to the formation of amyloid-like fibres over a period of many hours when the anion concentration exceeds a critical limit. Half of the amino acid residues are then organised in β-sheets. Both of the non-natively folded states of PGK contain more regular secondary structure than the native one. The misfolding starts in both cases from the acid-unfolded state, in which the molecules are essentially more expanded than in other denatured states, e.g. those effected by temperature or guanidine hydrochloride.

Introduction

“The native conformation of proteins is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment” (Anfinsen, 1973).

Variation of the environment should lead to a plasticity of the three-dimensional structure. The past history of the structure plays a role in determining non-equilibrium structures (metastable structures). Evidently, the two differently folded structures of the prion protein can coexist (Hornemann & Glockshuber, 1998). The environment of a given polypeptide segment may be provided by other segments of the polypeptide chain, other molecules, such as nucleic acids, polysaccharides or lipids, or various components of the solvent. Sudarsanam (1998) has shown by analysing the Protein Data Bank that there exists within the analysed 5420 protein structures numerous sequence-identical octa-, hepta-, and hexapeptide segments with different three-dimensional structures. Undoubtedly, their number will increase with the increasing number of elucidated protein structures Sudarsanam 1998, Kabsch and Sander 1984. This finding gives evidence that the structure of a polypeptide segment is determined not only by local interactions between neighbouring amino acid residues but also by non-local interactions. To examine the extent to which environmental factors influence the formation of secondary structural elements, Minor & Kim (1996) have designed an 11 amino acid residue sequence (dubbed the “chameleon” sequence) that folds as an α-helix when in one position but as a β-sheet when in another position of the sequence of the IgG-binding domain of the protein GB1. The chameleon sequence was designed to replace either α-helix residues 23–33 or β-sheet residues 42–52 of GB1. Recently, Davies et al. (1998) have shown experimentally that the feline leukaemia virus fusion peptide, consisting of 28 amino acid residues, can readily flip between random coil structure, α-helical and β-sheet conformation, depending upon its environment. The cell interior is not a homogeneous bag but consists of numerous compartments with different environments for protein molecules, such as the cytoplasm, ectosomes, lysosomes, membrane surfaces and the hydrophobic interior of membranes. It is an unanswered question, which conformational changes proteins may experience upon migrating from one compartment to another.

The environments in the different compartments can be simulated in vitro in particular cases. In this work, we are dealing with the question whether the above-described polymorphism of short polypeptide chains and segments can be observed also with long polypeptides. Structural plasticity of the prion polypeptide (231 amino acid residues) is known to cause prion-related diseases (Harrison et al., 1997).

Phosphoglycerate kinase from yeast (PGK) is a polypeptide consisting of 415 amino acid residues. Its three-dimensional structure has been determined under physiological conditions by Watson et al. (1982). PGK is a monomeric single-chain enzyme comprising two globular domains of about equal size. PGK is an α/β class protein; 35 % and 13 % of its amino acid residues are in α-helical and β-sheet conformation, respectively, under physiological conditions;. 27 % and 11 % of the amino acid residues forming the N-terminal domain are in α-helical and β-sheet conformation, respectively. The corresponding values for the C-terminal domain are 42 % and 14 %.

We will demonstrate that PGK can form two other folded conformations in addition to the native structure, one of which contains predominantly α-helices while the other has more β-structure than the native conformation. Both these conformations contain more secondary structure as compared to the native form. They are, however, less compact than the native molecule. Furthermore, this protein can adopt different random-walk conformations with varying coil dimensions caused by dissimilar non-local interactions of amino acid residues in different solvents. The average distributions of the φ,ψ-angles of the amino acid residues were determined for the acid-induced state of PGK (Damaschun et al., 1998).

Section snippets

Results

In this section, we first give a short summary of relevant data for native PGK and then describe the structural properties of acid-unfolded PGK, because this state of the protein at pH 2 is the starting conformation of two non-natively folded states of PGK, induced by addition of either TFE or salts.

Characterisation of native PGK

The CD spectra of native PGK in the far and near UV are shown in Figure 1(a) and (b), respectively. The hydrodynamic effective Stokes radius has been determined (Damaschun et al., 1993). From DLS measurements we obtained the translational diffusion coefficient D20,w0 = 7.21(±0.05) × 10−7 cm2/s and the corresponding Stokes radius RS = 2.97(±0.02) nm. The weak concentration dependence of D for PGK in 20 mM phosphate buffer (pH 6.5) can be expressed as D20,w = D20,w0 [1 + kDc] = D20,w0 [1 + (7.8 ×

Acid-induced unfolding of PGK

The CD spectra of PGK in 10 mM HCl (pH 2) in the far and near UV are shown in Figure 1(a) and (b), respectively. The far UV spectrum exhibits the characteristics of spectra for highly unfolded proteins. In the spectral range above 220 nm, it is very similar to the spectrum of GuHCl-unfolded PGK that is shown in Figure 1(a) for comparison. Moreover, the complete absence of the near UV spectrum (Figure 1(b)) is indicating the breakdown of any rigid tertiary structure.

Figure 2 shows the results of

The effect of TFE on acid-unfolded PGK

Addition of increasing amounts of TFE (5–50 %, v/v) to acid-unfolded PGK causes the formation of more and more helical secondary structure, as indicated by the CD data. Figure 5 shows the far-UV CD spectra for acid-denatured PGK and for PGK at pH 2 in the presence of 10, 20, 40 and 50 % (v/v) TFE. Evaluation of the secondary structure by the program CONTIN (Provencher & Glöckner, 1981) resulted in 76, 96 and 98 % helical structure for 20, 40 and 50 % (v/v) TFE, respectively.

In the near UV

The effect of anions on acid-unfolded PGK

Anion-induced partial refolding of acid-unfolded small globular proteins has been studied intensively by Fink and co-workers Goto et al 1990, Fink 1995, Fink et al 1997, Uversky et al 1998a, Uversky et al 1998b. The order of effectiveness of anions was shown to be ferricyanide > ferrocyanide > sulphate > trichloroacetate > thiocyanate > perchlorate > iodide > nitrate > trifluoroacetate > bromide > chloride (Goto et al., 1990). Moreover, different anions also induced different amounts of

Discussion

The native structure of PGK is stabilised by numerous non-local interactions between non-neighbouring amino acid residues. This structure can be destroyed by different external influences.

At temperatures below 4 °C and in the presence of 0.7 M GuHCl, the protein undergoes cold denaturation. Re-heating to 30 °C effects complete renaturation of the cold-denatured PGK, i.e. the molecule refolds to its native conformation. Cold-denatured PGK exhibits random coil conformation. The stiffness of the

Materials and methods

Yeast 3-phosphoglycerate kinase (EC 2.7.2.3) was purchased from Boehringer Mannheim GmbH (Germany), 2,2,2-trifluoroethanol 99 + % and the sodium salt of trichloroacetic acid 97 % from Sigma-Aldrich (Germany). All other chemicals were of analytical grade.

Acknowledgements

We thank R. Kröber for precise measurements. The authors are very grateful to U. Heinemann for critical reading of the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Da 292/6–2, He 1318/18–2) and by a grant from the Fonds der Chemischen Industrie to G.D.

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