REVIEW ARTICLE What does it mean to be natively unfolded? Vladimir N. Uversky 1 Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow, Russia; 2 Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA Natively unfolded or intrinsically unstructured proteins constitute a unique group of the protein kingdom. The evolutionary persistence of such proteins represents strong evidence in the favor of their importance and raises intriguing questions about the role of p rotein disorders in biological processes. Additionally, natively unfolded p ro- teins, with their lack of ordered structure, represent attractive targets for the biophysical studies of the unfolded p olypep- tide chain under physiological conditions in vitro.Thegoalof this study was to summarize the structural information on natively unfolded p roteins in o rder to evaluate their major conformational characteristics. It appeared that natively unfolded proteins are characterized by low overall hydro- phobicity and large net charge. They possess hydrodynamic properties typical of random coils in poor solvent, or pre- molten globule conformation. These proteins show a low level of ordered secon dary structure and no tightly packed core. They are very ¯exible, but may adopt relatively rigid conformations in the presence of natural ligands. Finally, in comparison with the globular proteins, natively unfolded polypeptides possess Ôturn outÕ responses to changes in the environment, as their structural complexities increase at high temperature or at e xtreme pH. Keywords: intrinsically unfolded protein; i ntrinsically disordered protein; unfolded protein; molten g lobule state; premolten globule state. WHAT ARE NATIVELY UNFOLDED PROTEINS? Before the phenomenon of natively unfolded p roteins will be considered, a de®nition of the major players is r equired. The importance of this issue follows from the fact that many proteins have been shown to have nonrigid structures under physiological conditions. These proteins may be separated in two d ifferent groups. Members of the ®rst group, despite their ¯ exibility, are rather compact and possess a well- developed secondary structure, i.e. t hey show properties typical o f the molten globule [1]. Proteins from the other group behave almost as random coils [2]. Only members of the second group will be described below. T hus, to b e considered as natively unfolded (or intrinsically unstruc- tured), a protein s hould be extremely ¯exible, essentially noncompact (extended), and have little or no ordered secondary structure under physiological conditions. WHY STUDY INTRINSICALLY DISORDERED PROTEINS? The number of p roteins and protein domains, that h ave been shown in vitro to have little or no ordered structure under physiological conditions, is rapidly increasing. In fact, over the past 1 0 years there has been an exponential increase in the number of such studies, starting from one paper in 1989, and ending with more than 30 in 2000. The current list of natively unfolded proteins includes more than 100 e ntries (91 of t hem were tabulated in our recent work [3]). This collection comprises the full-length proteins and their domains with chain length of more than 50 amino-acid residues. Including shorter polypeptides (30±50 residues long) would probably double this amount. The growing interest in this class of proteins is for several reasons. The ®rst issue is the structure±function relationship. The existence of biologically active but extremely ¯exible proteins questions the assumption that rigid well-folded 3D-structure is required for functioning. To o vercome this problem, it has been suggested that the lack of rigid globular structure under physiological conditions might represent a considerable functional a dvantage for Ônatively unfoldedÕ proteins, a s t heir large plasticity a llows them to interact ef®ciently with several d ifferent targets [4,5]. Moreover, a disorder/order transition induced in Ônatively unfoldedÕ proteins during the binding of speci®c targets in vivo might represent a simple me chanism for regulation of numerous cellular processes, i ncluding regulation of transcription and translation, and cell c ycle control. Precise contr ol o ver the thermodynamics of the binding process may also be achieved in this way (reviewed in [4,5]). E volutionary con tinuance of the intrinsically disordered proteins represents additional Correspondence to V. N. Uversky, Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064. Fax: + 831 459 2 935, Tel.: + 831 459 2915, E-mail: uversky@hydrogen.ucsc.edu Abbreviations:NAC,nonamyloidscomponent;AD,Alzheimer's disease; PD, Parkinson's disease; LB, Lewy body; LN, Lewy neurites; FTIR, Fourier-transform infrared; SAXS, small angle X-ray scatter- ing; R S , Stokes radius; N, native; MG, molten globule; PMG, pre- molten globule; U, unfolded; NU, natively unfolded. (Received 30 May 2001, revised 19 September 2001, accepted 31 October 2001) Eur. J. Biochem. 269, 2±12 (2002) Ó FEBS 2002 con®rmation of their importance and raises intriguing ques- tions on the role of protein disorder in biological processes. Secondly, biomedical aspects are of great importance too. It has been established t hat d eposition of some natively unfolded proteins is related to the development of several neurodegenerative disorders [6,7]. Examples include Alzheimer's disease [AD; deposition of amyloid-b,tau- protein, a-synu clein fragment nonamyloids component (NAC)] [8±11], Niemann-Pick disease type C, subacute sclerosing panencephalitis, a rgyrophilic grain d isease, myo- tonic dystrophy, motor neuron disease with neuro®brillary tangles (accumulation of tau-protein in the form of neuro- ®brillary tangles [10]), Down's syndrome (non®lamentous amyloid-b deposits [12]), Parkinson's disease (PD), demen- tia w ith Lewy b ody (LB), LB variant of AD, m ultiple system atrophy and Hallervorden-Spatz disease (deposition of a-synuclein in form of LBs and Lewy neurites (LNs) [13± 17]). Finally, intrinsically unstructured proteins represent a n attractive subject for the biophysical c haracterization of unfolded polypeptide chain under the physiolo gical condi- tions. The special term Ônatively unfoldedÕ was i ntroduced in 1994 to describe the behavior of tau protein [18], and has been frequently used ever since. Although large amounts of experimental data have been accumulated and several disordered proteins have been rathe r well characte rized (reviewed in [ 4,5]), the s ystematic analysis o f structural data for t he family of natively unfolded proteins has not been made as yet. This lack of methodical inspection of the conformational behavior of intrinsically unordered proteins has already lead to some confusion. For example, based on high thermostability, acidic pI, anomalous electrophoretic mobility, and t he high c ontent o f turns and random coil (% 50%), it w as concluded t hat m angan ese stabilizing protein is natively unfolded [19]. It was also suggested that the natively unfolded structure of this protein facilitates the highly effective protein±protein interactions that are neces- sary for its assembly into photosystem II. However, the validity of this conclusion was recently questioned [20]. In fact, more careful analysis of the structural properties of manganese stabilizing protein showed that it has a rather compact con formation w ith a well-developed secondary structure (47% bsheet), i.e. it i s closer t o a molten globule, than to an unfolded state [20]. Finally, it was reasonably noted that Ôthe structural feature of a Ônatively unfoldedÕ state is not the only possibility for conformation al ¯exibility of a protein to achieve optimal co nditions for interaction with other proteins. An alternative state with a high potential for structural adaptability is that of a mo lten globule' [20]. All this demonstrates that a s ystematic analysis of the structural and conformational properties of the family of natively unfolded proteins is required. WHY ARE INTRINSICALLY DISORDERED PROTEINS UNFOLDED? It is known that the unique three-dimensional structure of a globular protein is stabilized by various noncovalent interactions (conformational forces) of different nature, namely hydrogen bonds, hydrophobic interactions, van der Vaals interactions, e tc. F urthermore, a ll the n ecessary information for the correct folding o f a regular protein into the r igid biologically active conformation is included in i ts amino-acid sequence [21]. The a bsence of regular structure in natively unfolded proteins raises a question about the speci®c features of their amino-acid sequences. Some of the sequence peculiarities of these proteins were recognized long ago. These include the presence of numerous uncompen- sated charged groups (often negative), i.e. a large net charge at neutral pH, arising from the extreme pI values in such proteins [22±24], and a low content of hydrophobic amino- acid residues [22,23]. The comparison of the overall hydrophobicity and net charge of native and natively unfolded protein sequences showed that it is possible to predict whether a given amino- acid sequence encodes a native (folded ) or an intrinsically unstructured protein. In fact, this analysis established that the combination of low mean hydrophobicity and relatively high net charge r epresents an i mportant prerequisite for t he absence of compact structure in proteins under physiological conditions, t hus leading to Ônatively unfoldedÕ proteins [3]. Figure 1 represents the results of this survey and shows that the natively unfolded proteins are speci®cally localized within a unique r egion of the charge±hydrophobicity phase space. The solid line in this ® gure represents the border between intrinsically unstructured a nd nativeproteins. Ob viously, t his allows the estimation o f the ÔboundaryÕ mean hydrophobicity value, <H> b , below which a polypeptide chain with a given mean net charge <R> will be most probably unfolded: hHi b  hRi1X151 2X785 1 The v alidity of these predictions has been successfully shown f or sever al p roteins [ 25]. T his m eans that degree of compaction of a given polypeptide chain is determined by the balance in the competition between the charge repulsion driving unfolding and hydrophobic interactions driving folding. In an attempt to understand the relationship between sequence and disorder, Dunker a nd coauthors have elabo- rated several neuronal network predictors [5,26±35]. They assumed that if a protein structure has evolved to have a functional disordered s tate, then a propensity for disorder might b e predictable from its amino-acid sequence a nd composition. The results of such analysis were more than impressive. It h as been established that disordered r egions share at least some common sequence features over many proteins. This includes low sequence complexity, with amino- acid compositional bias and high predicted ¯exibility [28,29]. Furthermore, the majority of the intrinsically disord ered proteins, being substantially depleted in I, L, V, W, F, Y, C, and N, a re enriched in E, K, R, G, Q, S, P, and A [5]. Note that these f eatures may account for the low o verall hydro- phobicity and high net charge of the polypetide c hain of natively unfolded proteins. Interestingly, more than 15 000 proteins in the SwissProt database were identi®ed a s having long regions of sequence that share these same features [31]. WHAT ARE THE GENERAL STRUCTURAL CHARACTERISTICS OF NATIVELY UNFOLDED PROTEINS? The general conformational properties of intrinsically unfolded proteins are summarized below. Here we will mostly focus on the structural characteristics, which m ake Ó FEBS 2002 Natively unfolded proteins (Eur. J. Biochem. 269)3 such proteins exceptional among others. These a re low compactness, absence of globularity, low secondary struc- ture content, and high ¯exibility. Compactness The most unambiguous characteristic of the conformational state of a globular protein is t he hydrodynamic dimensions. It was noted long ago that h ydrodynamic techniques may help to r ecognize when a protein has lost all of i ts noncovalent structure, i.e. when it b ecame unfolded [2]. This is because an essential increase in the hydro dynamic volume is associated with the unfolding of a protein molecule. I t is known that globular proteins may exist in at least four different conformations, native, molten globule, premolten g lobule a nd unfolded [1,36±39], that may easily be discriminated by the degree of compactness of the polypeptide chain. Finally, it has been established that t he native and unfolded c onformations of globular pr oteins possess very different molecular mass dependencies of their hydrodynamic radii (the Stokes radius), R S [2,40,41]. In order to clarify the physical nature of natively unfolded proteins, Fig. 2 compares log(R S )vs.log(M) curves for these proteins (see Table 1 for details) with same d epen- dencies for the native, molten globule, premolten globule, and urea- or GdmCl-unfolded globular proteins (data for different conformations of globular proteins were taken from [42]). The log(R S )vs.log(M) dependencies for different conformations of globular proteins might be described by straight lines: logR N S À0X204Æ0X0230X357Æ0X005ÁlogM2 logR MG S À0X053 Æ 0X0940X 334 Æ 0X021ÁlogM 3 logR PMG S À0X21 Æ 0X180X392 Æ 0X041ÁlogM 4 logR Uurea S À0X649 Æ 0X0160X521 Æ 0X004ÁlogM 5 logR UGdmCl S À0X723 Æ0X0330X543Æ0X007ÁlogM 6 Where N, native; MG, molten globule; PMG, premolten globule a nd U(urea) and U(GdmCl) correspond to the unfolded urea and GdmCl globular proteins, r espectively. As for natively unfolded proteins, Fig. 2 clearly shows that in respect of the ir log(R S ) vs. log(M) dependence they may be divided in two groups (see Table 1). One group of the i ntrinsically unstructured proteins behaves as random coils in poor solvent [denoted as natively unfolded (NU)(coil)]. Proteins from the other group are essentially more compact, being c lose with respect to their hydrody- namic characteristics to premolten globules [denoted as NU(PMG)]: logR NUcoil S À0X551 Æ 0X0320X493 Æ 0X008ÁlogM 7 logR NUPMG S À0X239 Æ0X0550X403Æ0X 012ÁlogM 8 This is a very important obse rvation, whic h may help in understanding the physical natu re of the natively unfolded proteins. In fact, it is well established that the behavior of unfolded proteins obeys the theoretical and empirical rules that apply to linear random coils [1]. Speci®cally, it is known that the hydrodynamic dimensions of random coils depends Mean hydrophobicity 0.1 0.2 0.3 0.4 0.5 0.6 Mean net charge 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Fig. 1. Comparison of the mean net charge and the mean hydrophobicity for a set of 275 folded (open circles) and 105 na tivel y unfolded proteins (gra y circles). The solid line represents the border between intrinsically unstructured and native proteins (see text). Part of the data for this plot is taken from [3]. 4 V. N. Uversky (Eur. J. Biochem. 269) Ó FEBS 2002 essentially on the quality of solvent [2,40,43]. A poor solvent encourages the attraction of macromolecular segments a nd hence a chain has to squeeze. Whereas, in a good solvent, repulsive forces act primarily between the segments a nd the macromolecule conforms to a loose ¯uctuating c oil [44]. Water is a poor solvent, whereas solutions of urea and GdmCl are rather good solvents, w ith GdmCl being closer to the ideal one [2,40]. This difference in solvent quality may account for the observed divergence in log(R S )vs.log(M) dependencies for the coil-like part o f intrinsically unstruc- tured proteins. The existence of well-de®ned difference between the log(R S ) vs. log(M) dependencies for globular proteins unfolded by urea and GdmCl also should be noted in this respect. Globularity Another v ery important structural parameter i s the degree of globularization that re¯ects the p resence or absence of tightly packed core in the protein molecule. I n f act, it has been shown that the protein molecules in PMG are characterized b y low (coil-like) intramolecular packing density [ 37,38,42,45]. This information could be extracted from the analysis of small angle X-ray scattering (SAXS) data (Kratky plot), whose shape is sensitive to the conformational state of the scattering protein molecules [45±48]. It has been shown that a scattering curve in the Kratky plot has a characteristic maximum when the globular protein is i n the native state o r in the molten globule state (i.e. has a globular structure). If a protein is completely unfolded or in a premolten globule con- formation ( has n o g lobular structure), such a maximum will be absent on the r espective scattering curve [37,38,42, 45±48]. Figure 3A compares the Kratky plots of three natively unfolded p roteins (a-syn uclein, prothymosin a and c aldes- mon 636±771 fragment) with t hat of t he rigid g lobular protein SNase. One can s ee that intrinsically unstructured proteins give Kratky plots without m axima typical of folded conformations of globular proteins. The same d ata has also been reported f or another i ntrinsically unordered protein, pig calpastatin domain I [49]. Thus, t hese four natively unfolded proteins are characterized by the absence of globular structure, or, in other words, they do not have a tightly packed core under physiological conditions in vitro. This is a very important observation, which allows the assumption that all other natively unfolded proteins may possess the same property. In fact, the analysis of hydrodynamic data s hows t hat two of the three consid ered proteins (a-synuclein and prothymosin a) behave as coils in poor solvent, whereas R S of caldesmon 636±771 fragment is typical of PMG (see Table 1 ). Consequently, r epresen- tatives of both classes of intrinsically unstructured proteins (coil-like and PMG-like) have been shown to b e charac- terized by the absence of rigid globular core. This i s i n goodagreementwithSAXSdataonconformational characteristics of t he PMG state of globular proteins [37,38,42,45]. Secondary structure Figure 3B presents the far-UV CD s pectra of a-synuclein, prothymosin a, phosphodiesterase c-subunit and caldes- mon 636±771 fragment as typical representatives of the log (M) 3.5 4.0 4.5 5.0 5.5 log ( R S ) 1.0 1.5 2.0 Fig. 2. Dependencies of the hydrodynamic dime nsions, R S , on protein molecular mass, M, for native (gray circles), molten globule (gray reversed triangles), premolten globule (gray squares), 8 M urea-unfolded (gray diamonds) and 6 M GdmCl-unfolded (gray triangles) conformational states of globular proteins and natively unfolded proteins with coil-like (open circles) and PMG-like properties (open reversed triangles). Thedatausedtoplot dependencies for native, molten globu le, premolten glo bule a nd GdmCl-unfolded states of globular proteins are taken from [42]. T he data for natively unfolded p roteins and u rea-unfolded conformation of globular proteins are s umma rized in T ables 1 and 2, respectively. Dashed lines represent least square ®ts of data earlier obtained for native and urea- or GdmCl-unfolded globular proteins [41]. Ó FEBS 2002 Natively unfolded proteins (Eur. J. Biochem. 269)5 family of natively unfolded p roteins. One can see that these proteins (as well as all other i ntrinsically unstructured proteins, whose far-UV CD spectra were studied) possess distinctive far-UV CD s pectra with characteristic deep minima in vicinity of 200 nm, and relatively low ellipticity at 220 nm. The analysis of these spectra yields low content of ordered secondary structure (a helices and b sheets). This is also con®rmed b y the Fourier-transform infrared (FTIR) analysis of secondary structure composition of natively unfold ed proteins, such as tau protein [18], a- synuclein [24,50], b-andc-synucleins; a s -casein [51], and cAMP-dependent protein kinase inhibitor [ 52]. Important- ly, even the caldesmon 636 ±771 fra gment, w hich wa s shown to have hydrodynamic properties typical of the PMG (see above), posse sses far-UV CD characteristic of essentially distorted polypeptide chain. Thus, the low overall content of ordered secondary structure could be considered as a general property of intrinsically unstruc- tured p roteins. High ¯exibility The fact that intrinsically unfolded proteins are character- ized by an increased intramolecular ¯exibility may be easily derived from a large a mount of NMR studies (summarized in [4,5,53]). Moreover, recent advances in NMR technology (especially the use of heteronuclear multidimensional approach) have even opened the way to detailed structural and dynamic description o f t hese proteins [4]. Increased ¯exibility o f n atively unfo lded proteins is i ndirectly con- ®rmed by their extremely h igh s ensitivity to protease degradation in vit ro [4,5,54±59]. Table 1. Hydrodynamic characteristics of the natively unfolded proteins. M r (kDa) R S (A Ê ) Reference Coil-like proteins a-Fetoprotein, 447±480 fragment 3.6 15.5 [85] Vmw65 C-terminal domain 9.3 28 [86] PDE c 9.7 26 E m protein 11.2 28.2 [87] Apo-cytochrome c 11.7 30 [88] Prothymosin a 12.1 24.3 [62] Fibronectin binding domain B 12.3 30.7 [89] c-Synuclein 13.3 30.4 Fibronectin binding domain A 13.7 31.7 [89] Ribonuclease A, reduced 13.7 50.6 [41] b-Synuclein 14.3 32 [90] a-Synuclein 14.5 32.3 [24,50] Fibronectin binding domain D 14.7 31.8 [89] Stathmin 17 33 [91] CFos-AD domain, 216±380 fragment 17.3 35 [92] Calf thymus histone 19.8 36.7 [1] b-Casein 24 41.7 [1] Phosvitin 24.9 39.9 [1] Chromatogranin A 48.3 58.5 [76] Caldesmon 140 91 [93] MAP-2 220 122 [94] PMG-like proteins Osteocalcin 5.4 18.4 [73] Heat stable protein kinase inhibitor 7.9 22.3 [52] Caldesmon 636±771 fragment 14 28.1 SNaseD, A90S mutant 14.1 25 [95] Pf1 gene 5 protein, 1±144 fragment (D4 domain) 15.8 29.5 [96] PPI-1 20.8 32.3 [97] DARRP-32 23.1 34 [22] Manganese stabilizing protein, L245E mutant 26.5 32.7 [98] Calreticulin, human )41C fragment 40.6 46.2 [59] Calsequestrin, rabbit 45.2 45 [99] Calreticulin, huiman 46.8 46.2 [59] Calreticulin, bovine 47.6 44.2 [59] Taka-amylase A, reduced 52.5 43.1 [1] SdrD protein, B1-B5 fragment 64.8 54.7 [75] Chromatogranin B 77.3 50.3 [77] Topoisomerase I 90.7 58.5 [100] Fibronectin 530 115 [101] 6 V. N. Uversky (Eur. J. Biochem. 269) Ó FEBS 2002 ENVIRONMENTAL INFLUENCES ON THE NATIVELY UNFOLDED PROTEINS Temperature effects Figure 4A depicts temperature-induced changes i n the far- UV CD spectra of a-synuclein [50] measured at different temperatures. At low temperatures, the protein shows a far- UV CD spectrum typical of an unfolded polypeptide chain. As the t emperature is increased, the spectrum changes, consistent with temperature-induced formation of second- ary structure. Figure 4 B represents the temperature-depen- dence of [h] 222 for a-synuclein, caldesmon 636±771 fragment, and phosphodiesterase c-subunit. One can see that for these three proteins major spectral changes occur over the range of 3 to 30±50 °C. Further heating leads to a less pronounced effects. Analogous temperature dependen- cies indicative of heat-induced str ucture formation have been reported for the receptor extracellular domain of nerve growth factor [60] and a s -casein [61]. Interestingly, it has been shown that the structural changes induced in all these proteins by heating are completely reversible. Thus, an increase in temperature induces the partial folding o f intrinsically unstructured proteins, rather than the unfolding typical o f g lobular proteins. The effects of elevated temper- ature may be attributed to increased strength of the hydrophobic interaction at higher temperatures, leading to a stronger hydrophobic driving force for folding. This observation de®nitely has t o be t aken into account while discussing conformational behavior of intrinsically unstructured proteins. Effect of pH Figure 4C represents the pH dependence o f [ h] 222 for a-synu clein and prothymosin a. There is little change i n the far-UV CD spectra between pH % 9.0 and % 5.5. However, a decrease in pH from 5.5 to 3.0 results in a substantial increase in negative intensity in the vicinity of 220 nm. It has also been established that the pH-induced changes in the far-UV CD spectrum of these t wo proteins were completely reversible and consistent with the forma- tion of partially folded PMG-like intermediate conforma- tion [50,62]. Same pH-induced structural transformations have been described for pig calpastatin domain I [39], histidine rich protein I I [63], a nd the naturally occurring human peptide LL-37 [64]. T hese observations show that a decrease (or increase) in pH induces partial folding of intrinsically unordered proteins due to the minimization of their large net charge present at neutral pH, thereby decreasing charge/charge intramolecular repulsion and permitting hydrophobic-driven collapse to the partially folded inter- mediate. Effect of counter ions It was already noted t hat, under physiological pH, intrin- sically unstructured proteins are unfolded mainly because of the electrostatic repulsion between the noncompensated charges of the same sign. To some extent, this resembles the Fig. 3. Conformational characteristics o f intrinsically disordered pro- teins. (A) Kratky plots of SAXS data for natively unfolded a-synuclein (1), prothymosin a (2) a nd caldesmon 636±771 fragment (3). The Kratky plot of native globular SNase is shown for comparison (4). (B) Far-UV CD spectra of intrinsically unordered proteins, a-synuclein (1), prothymosin a (2), caldesmon 636±771 fragment (3) and phos- phodiesterase c-subunit (4). Table 2. Hydrodynamic characteristics of 8 M urea-unfolded p ro teins without cross-links. Protein M r (kDa) R S (A Ê ) Reference Insulin 3 14.6 [41] Ubiquitin 8.5 24.6 Cytochrome c 11.7 4.05 Ribonuclease A 13.7 32.4 [41] Lysozyme 14.2 33.1 [41] Hemoglobin 15.5 33.5 Myoglobin 16.9 35.1 b-Lactoglobulin 18.5 37.8 [41] Chymotrypsinogen 25.7 45 [41] Carbonic anhydrase B 28.8 47.8 [41] b-Lactamase 28.8 48.9 [41] Ovalbumin 43.5 58.8 Serum albumin 66.3 74 [41] Lactate dehydrogenase 35.3 52 GAP dehydrogenase 36.3 54 Aldolase 40 57 Transferrin 81 81 Thyroglobulin 165 116 Ó FEBS 2002 Natively unfolded proteins (Eur. J. Biochem. 269)7 situation occurring for many proteins at e xtremely low o r high pH. It has been established that these unfolded proteins could be transformed into more ordered conformations if electrostatic repulsion was reduced by binding of oppositely charged ions [65,66]. Similar s ituation may be expected for natively unfolded proteins, and, in fact, the metal i on- stimulated conformational changes have been described for many intrinsically unstructured proteins. As an illustration, Fig. 4D represents the [h] 222 depen- dencies on [ Al 3+ ]fora-synuclein. One can s ee that an increase in the cation content is accompanied by an essential increase in the intensity of the far-UV CD spectra, re¯ecting partial folding of the protein. It has been established that other cations (monovalent, bivalent and trivalent) induce conformational changes in a-synuclein and transform this natively unfolded protein into a partially folded intermedi- ate too. The folding strength of cations increases with the ionic charge density incre ase [67]. This re¯ects t he effective screening of the Coulombic charge/charge repulsion. For polyvalent c ations, an additional important factor could b e hypothesized, which is the potential capability for cross- linking or bridging between two or more carboxylates. Importantly, human antibacterial protein LL-37, a natively unfolded p rotein with extremely basic net charge, was shown to be essentially folded in the presence of several anions [64]. WHAT ELSE IS REQUIRED FOR INTRINSICALLY UNORDERED PROTEINS TO FOLD? Structure forming role of natural ligands It has been suggested that natively unfolded proteins may be signi®cantly folded in their normal cellular milieu due to binding to speci®c targets and ligands (such a s a variety of small molecules, s ubstrates, cofactors, other proteins, nucleic acids, membranes, etc.) [3±5,53,68]. The structure- forming effect of natural partners can be explained by their in¯uence o n the m ean hydrophobicity and/or net charge of the natively unfolded polypeptide. In fact, any interaction of such protein with natural ligand affecting mean net c harge and/or mean hydrophobicity of the protein could c hange t hese parameters in such a way that they will approach values typical of folded native proteins. This hypothesis has been con®rmed by calculation the joint mean net charge and mean hydrophobicity of complexes of several natively unfolded p roteins, ostecalcin, Fig. 4. Eect of environmental factors on conformational properties of natively unfolded proteins. (A) Heating-induced secondary structure formation in the n atively unfolded a-synuclein. Representative f ar-UV CD s pectra of the protein measured at dierent temperatures. (B) Temperature- induced changes in far-UV CD spectrum ([h] 222 vs. temperature depen dence) measured for a-synuc lein (triangles), phosphodiesterase c-subunit (squares), and caldesmon 636±771 fragment (circles). (C) pH-induced structure formation ([h] 222 vs. pH dependence) in the natively unfolded a-synuclein (circles) and prothymosin a (triangles). (D) Cation-induced structure formation in natively unfolded a-synuclein. Data for a-synuclein and protymosin a are taken from [50,67] and [62], respectively. 8 V. N. Uversky (Eur. J. Biochem. 269) Ó FEBS 2002 osteonectin, a-casein, HPV16 E7 protein, calsequestrin, manganese s tabilizing p rotein and HIV-1 integrase, with their n atural ligands, metal ions [3]. The e xistence of pronounced ligand-induced folding has been indeed established in numerous in vitro studies for many intrin- sically unstructured proteins. E xamples include: DNA (or RNA) induced structure f ormation in protamines [69,70], Max protein [57], high mobility group proteins HMG-14 [71] and HMG-17 [72]; cation-induced folding o f o stecal- cine [73], osteonectine [ 74], S drd protein [75], chromatog- ranins A [ 76] and B [77], D131D fragment of SNase [78], histone H1 [79], protamine [70] and prothymosin-a [80]; folding of cytochrome c inthepresenceofheme[81]; membrane-induced secondary structure formation in para- thyroid hormone related protein [82]; trimethylamine N-oxide induced structure formation in glucocorticoid receptor [83]; h eme-induced folding of histidine-rich pro- tein II [84], and many others. 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