Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA Natively unfolded or intrinsically unstructured proteins constitute a unique group of the prote
Trang 1Eur J Biochem 269, 2-12 (2002) © FEBS 2002
REVIEW ARTICLE
What does it mean to be natively unfolded?
Vladimir N Uversky
‘Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow, Russia;
? 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 protein disorders in
biological processes Additionally, natively unfolded pro-
teins, with their lack of ordered structure, represent attractive
targets for the biophysical studies of the unfolded polypep-
tide chain under physiological conditions in vitro The goal of
this study was to summarize the structural information on
natively unfolded proteins in order 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 secondary structure and no tightly packed core They are very flexible, but may adopt relatively rigid conformations in the presence of natural ligands Finally, in comparison with the globular proteins, natively unfolded polypeptides possess ‘turn out’ responses to changes in the environment, as their structural complexities increase at high
temperature or at extreme pH
Keywords: intrinsically unfolded protein; intrinsically disordered protein; unfolded protein; molten globule state; premolten globule state
WHAT ARE NATIVELY UNFOLDED
PROTEINS?
Before the phenomenon of natively unfolded proteins will
be considered, a definition of the major players is required
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 different groups Members of the first group, despite
their flexibility, are rather compact and possess a well-
developed secondary structure, i.e they show properties
typical of 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 Thus, to be
considered as natively unfolded (or intrinsically unstruc-
tured), a protein should be extremely flexible, essentially
noncompact (extended), and have little or no ordered
secondary structure under physiological conditions
Correspondence to V N Uversky, Department of Chemistry and
Biochemistry, University of California, Santa Cruz, CA 95064
Fax: + 831 459 2935, Tel.: + 831 459 2915,
E-mail: uversky@hydrogen.ucsc.edu
Abbreviations: NAC, nonamyloids component; 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; Rs, 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)
WHY STUDY INTRINSICALLY DISORDERED PROTEINS?
The number of proteins and protein domains, that have been shown in vitro to have little or no ordered structure under physiological conditions, is rapidly increasing In fact, over the past 10 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 entries (91 of them 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 first issue is the structure—function relationship The existence of biologically active but extremely flexible proteins questions the assumption that rigid well-folded 3D-structure is required for functioning To overcome this problem, it has been suggested that the lack of rigid globular structure under physiological conditions might represent a considerable functional advantage for ‘natively unfolded’ proteins, as their large plasticity allows them to interact
efficiently with several different targets [4,5] Moreover, a
disorder/order transition induced in ‘natively unfolded’ proteins during the binding of specific targets in vivo might represent a simple mechanism for regulation of numerous cellular processes, including regulation of transcription and translation, and cell cycle control Precise control over the thermodynamics of the binding process may also be achieved
in this way (reviewed in [4,5]) Evolutionary continuance of the intrinsically disordered proteins represents additional
Trang 2confirmation 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 that deposition 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, o-synuclein fragment nonamyloids component
(NAC)] [8-11], Niemann-Pick disease type C, subacute
sclerosing panencephalitis, argyrophilic grain disease, myo-
tonic dystrophy, motor neuron disease with neurofibrillary
tangles (accumulation of tau-protein in the form of neuro-
fibrillary tangles [10]), Down’s syndrome (nonfilamentous
amyloid-B deposits [12]), Parkinson’s disease (PD), demen-
tia with Lewy body (LB), LB variant of AD, multiple
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 an
attractive subject for the biophysical characterization of
unfolded polypeptide chain under the physiological condi-
tions
The special term ‘natively unfolded’ was introduced 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 rather well characterized
(reviewed in [4,5]), the systematic analysis of structural data
for the 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 the high content of turns and random coil
(~ 50%), it was concluded that manganese 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 conformation with a well-developed secondary
structure (47% sheet), i.e it is closer to a molten globule,
than to an unfolded state [20] Finally, it was reasonably
noted that ‘the structural feature of a ‘natively unfolded’ state
is not the only possibility for conformational flexibility of a
protein to achieve optimal conditions for interaction with
other proteins An alternative state with a high potential for
structural adaptability is that of a molten globule’ [20]
All this demonstrates that a systematic 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, etc Furthermore, all the necessary
information for the correct folding of a regular protein into
the rigid biologically active conformation is included in its amino-acid sequence [21] The absence of regular structure
in natively unfolded proteins raises a question about the specific 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 represents an important prerequisite for the absence of compact structure in proteins under physiological conditions, thus leading to ‘natively unfolded’ proteins [3] Figure | represents the results of this survey and shows that the natively unfolded proteins are specifically localized within
a unique region of the charge—hydrophobicity phase space The solid line in this figure represents the border between intrinsically unstructured and native proteins Obviously, this allows the estimation of the ‘boundary’ mean hydrophobicity value, < H >, below which a polypeptide chain with a given mean net charge < R> will be most probably unfolded:
The validity of these predictions has been successfully shown for several proteins [25] This means 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 and 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 state, then a propensity for disorder might be predictable from its amino-acid sequence and composition The results of such analysis were more than impressive It has been established that disordered regions share at least some common sequence features over many proteins This includes low sequence complexity, with amino- acid compositional bias and high predicted flexibility [28,29] Furthermore, the majority of the intrinsically disordered
proteins, being substantially depleted in I, L, V, W, F, Y, C, and N, are enriched in E, K, R, G, Q, S, P, and A [5] Note
that these features may account for the low overall hydro- phobicity and high net charge of the polypetide chain of natively unfolded proteins Interestingly, more than 15 000 proteins in the SwissProt database were identified as 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 make
Trang 3
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Mean hydrophobicity Fig 1 Comparison of the mean net charge and the mean hydrophobicity for a set of 275 folded (open circles) and 105 natively unfolded proteins (gray 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]
such proteins exceptional among others These are low
compactness, absence of globularity, low secondary struc-
ture content, and high flexibility
Compactness
The most unambiguous characteristic of the conformational
state of a globular protein is the hydrodynamic dimensions
It was noted long ago that hydrodynamic techniques may
help to recognize when a protein has lost all of its
noncovalent structure, ic when it became unfolded [2]
This is because an essential increase in the hydrodynamic
volume is associated with the unfolding of a protein
molecule It is known that globular proteins may exist in
at least four different conformations, native, molten globule,
premolten globule and unfolded [1,36—39], that may easily
be discriminated by the degree of compactness of the
polypeptide chain Finally, it has been established that the
native and unfolded conformations of globular proteins
possess very different molecular mass dependencies of their
hydrodynamic radii (the Stokes radius), Rs [2,40,41]
In order to clarify the physical nature of natively unfolded
proteins, Fig 2 compares log(Rs) vs log(M) curves for
these proteins (see Table | for details) with same depen-
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( Rs) vs log(/) dependencies for different
conformations of globular proteins might be described by
straight lines:
log(RY) = —(0.204 £0.023) + (0.35740.005)-log(M) (2)
log(R¥S) = —(0.053 + 0.094) + (0.334 + 0.021) - log(M)
(3)
log(REYS) = —(0.21 + 0.18) + (0.392 + 0.041) - log(M)
(4)
(urea)
log(Re"") = —(0.649 £ 0.016) + (0.521 £0.004) -log(M)
(5) log(RYC™) — — (0.723 40.033) + (0.543 +0.007) -log(M)
(6)
Where N, native; MG, molten globule; PMG, premolten
globule and U(urea) and U(GdmCl) correspond to the unfolded urea and GdmCl globular proteins, respectively
As for natively unfolded proteins, Fig 2 clearly shows that in respect of their log(Rs) vs log(M) dependence they may be divided in two groups (see Table 1) One group of the intrinsically 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 close with respect to their hydrody- namic characteristics to premolten globules [denoted as NU(PMG)]:
log(REV&") = —(0.551 40.032) + (0.493 + 0.008) -log(M)
(7)
log(RNUPM) = — (0.239 £0.055) + (0.403 £0.012) -log(M)
(8)
This is a very important observation, which may help in understanding the physical nature 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] Specifically, it is known that the hydrodynamic dimensions of random coils depends
Trang 4
log (M) Fig 2 Dependencies of the hydrodynamic dimensions, Rs, 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) The data used to plot dependencies for native, molten globule, premolten globule and GdmCl-unfolded states of globular proteins are taken from [42] The data for natively unfolded proteins and urea-unfolded conformation of globular proteins are summarized in Tables 1 and 2, respectively Dashed lines represent least square fits of data earlier obtained for native and urea- or GdmCl-unfolded globular proteins [41]
essentially on the quality of solvent [2,40,43] A poor solvent
encourages the attraction of macromolecular segments and
hence a chain has to squeeze Whereas, in a good solvent,
repulsive forces act primarily between the segments and the
macromolecule conforms to a loose fluctuating coil [44]
Water is a poor solvent, whereas solutions of urea and
GdmCl are rather good solvents, with GdmCl being closer
to the ideal one [2,40] This difference in solvent quality may
account for the observed divergence in log(Rs) vs log(M)
dependencies for the coil-like part of intrinsically unstruc-
tured proteins The existence of well-defined difference
between the log(Rs) vs log(M) dependencies for globular
proteins unfolded by urea and GdmC_] also should be noted
in this respect
Globularity
Another very important structural parameter is the degree
of globularization that reflects the presence or absence of
tightly packed core in the protein molecule In fact, it has
been shown that the protein molecules in PMG are
characterized by 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 in the native state or 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 no globular structure), such a maximum
will be absent on the respective scattering curve [37,38,42,
45-48]
Figure 3A compares the Kratky plots of three natively unfolded proteins (a-synuclein, prothymosin « and caldes- mon 636-771 fragment) with that of the rigid globular protein SNase One can see that intrinsically unstructured proteins give Kratky plots without maxima typical of folded conformations of globular proteins The same data has also been reported for another intrinsically unordered protein, pig calpastatin domain I [49] Thus, these 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 shows that two of the three considered proteins (a-synuclein and prothymosin «) behave as coils in
poor solvent, whereas Rg of caldesmon 636-771 fragment
is typical of PMG (see Table 1) Consequently, represen- tatives of both classes of intrinsically unstructured proteins (coil-like and PMG-like) have been shown to be charac- terized by the absence of rigid globular core This is in good agreement with SAXS data on conformational characteristics of the PMG state of globular proteins [37,38,42,45]
Secondary structure Figure 3B presents the far-UV CD spectra of o-synuclein, prothymosin «, phosphodiesterase y-subunit and caldes- mon 636-771 fragment as typical representatives of the
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Table 1 Hydrodynamic characteristics of the natively unfolded proteins
M, (kDa) Rs (A) Reference Coil-like proteins
a-Fetoprotein, 447-480 fragment 3.6 15.5 [85]
Vmw65 C-terminal domain 9.3 28 [86]
Fibronectin binding domain B 12.3 30.7 [89]
Fibronectin binding domain A 13.7 31.7 [89]
Ribonuclease A, reduced 13.7 50.6 [41]
Fibronectin binding domain D 14.7 31.8 [89]
CFos-AD domain, 216-380 fragment 17.3 35 [92]
Calf thymus histone 19.8 36.7 [1]
PMG-like proteins
Heat stable protein kinase inhibitor 7.9 22.3 [52]
Caldesmon 636-771 fragment 14 28.1
SNaseD, A908 mutant 14.1 25 [95]
Pfl gene 5 protein, 1-144 fragment (D4 domain) 15.8 29.5 [96]
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]
family of natively unfolded proteins One can see that these
proteins (as well as all other intrinsically unstructured
proteins, whose far-UV CD spectra were studied) possess
distinctive far-UV CD spectra 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 f sheets)
This is also confirmed by the Fourier-transform infrared
(FTIR) analysis of secondary structure composition of
natively unfolded proteins, such as tau protein [18], œ-
synuclein [24,50], B- and y-synucleins; «,-casein [51], and
cAMP-dependent protein kinase inhibitor [52] Important-
ly, even the caldesmon 636-771 fragment, which was
shown to have hydrodynamic properties typical of the
PMG (see above), possesses 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 proteins
High flexibility
The fact that intrinsically unfolded proteins are character- ized by an increased intramolecular flexibility may be easily derived from a large amount 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 of these proteins [4] Increased flexibility of natively unfolded proteins is indirectly con- firmed by their extremely high sensitivity to protease degradation in vitro [4,5,54-59].
Trang 6
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Fig 3 Conformational characteristics of intrinsically disordered pro-
teins (A) Kratky plots of SAXS data for natively unfolded o-synuclein
(1), prothymosin « (2) and 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, o-synuclein
(1), prothymosin « (2), caldesmon 636-771 fragment (3) and phos-
phodiesterase y-subunit (4)
ENVIRONMENTAL INFLUENCES
ON THE NATIVELY UNFOLDED
PROTEINS
Temperature effects
Figure 4A depicts temperature-induced changes In the Íar-
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 temperature is increased, the spectrum changes,
consistent with temperature-induced formation of second-
ary structure Figure 4B represents the temperature-depen-
fragment, and phosphodiesterase y-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-
cles indicative of heat-induced structure formation have
been reported for the receptor extracellular domain of nerve
growth factor [60] and «,-casein [61] Interestingly, it has
been shown that the structural changes induced 1n all these
proteins by heating are completely reversible Thus, an
increase 1n temperature induces the partial folding of
Table 2 Hydrodynamic characteristics of 8 mM urea-unfolded proteins without cross-links
Protein M,.(kDa) Rs (A) 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 46.9 [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
intrinsically unstructured proteins, rather than the unfolding typical of globular 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 definitely has to be taken into account while discussing conformational behavior of intrinsically unstructured proteins
Effect of pH Figure 4C represents the pH dependence of [6]> for a-synuclein and prothymosin o There is little change in 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 1n the far-UV CD spectrum of these two 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 [63], and the naturally occurring human peptide LL-37 [64] These observations show that a decrease (or increase) 1n 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 that, 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
Trang 78 V N Uversky (Eur J Biochem 269)
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Temperature (°C) [Cation] (mM) Fig 4 Effect of environmental factors on conformational properties of natively unfolded proteins (A) Heating-induced secondary structure formation
in the natively unfolded o-synuclein Representative far-UV CD spectra of the protein measured at different temperatures (B) Temperature- induced changes in far-UV CD spectrum ([6]222 vs temperature dependence) measured for o-synuclein (triangles), phosphodiesterase y-subunit (squares), and caldesmon 636-771 fragment (circles) (C) pH-induced structure formation ([@]222 vs pH dependence) in the natively unfolded a-synuclein (circles) and prothymosin « (triangles) (D) Cation-induced structure formation in natively unfolded o-synuclein Data for z-synuclein and protymosin « are taken from [50,67] and [62], respectively
situation occurring for many proteins at extremely low or
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 situation may be expected for
natively unfolded proteins, and, in fact, the metal 1on-
stimulated conformational changes have been described for
many intrinsically unstructured proteins
As an illustration, Fig 4D represents the [6] depen-
dencies on [AI””] for o-synuclein One can see that an
increase 1n the cation content 1s accompanied by an essential
increase 1n the intensity of the far-UV CD spectra, reflecting
partial folding of the protein It has been established that
other cations (monovalent, bivalent and trivalent) induce
conformational changes in o-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 increase [67] This reflects the effective
screening of the Coulombic charge/charge repulsion For
polyvalent cations, an additional important factor could be
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 protein 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 significantly folded in their normal cellular milieu due
to binding to specific targets and ligands (such as a variety
of small molecules, substrates, cofactors, other proteins, nucleic acids, membranes, etc.) [3—5,53,68] The structure-
forming effect of natural partners can be explained by their influence on the mean hydrophobicity and/or net charge of the natively unfolded polypeptide In fact, any interaction of such protein with natural ligand affecting mean net charge and/or mean hydrophobicity of the protein could change these parameters in such a way that they will approach values typical of folded native proteins This hypothesis has been confirmed by calculation the joint mean net charge and mean hydrophobicity of complexes of several natively unfolded proteins, ostecalcin,
Trang 8osteonectin, o-casein, HPV16 E7 protein, calsequestrin,
manganese stabilizing protein and HIV-1 integrase, with
their natural ligands, metal ions [3] The existence of
pronounced ligand-induced folding has been indeed
established in numerous in vitro studies for many intrin-
sically unstructured proteins Examples include: DNA (or
RNA) induced structure formation in protamines [69,70],
Max protein [57], high mobility group proteins HMG-14
[71] and HMG-17 [72]; cation-induced folding of ostecal-
cine [73], osteonectine [74], Sdrd protein [75], chromatog-
ranins A [76] and B [77], A131A fragment of SNase [78],
histone H1 [79], protamine [70] and prothymosin-« [80];
folding of cytochrome c in the presence of heme [81];
membrane-induced secondary structure formation in para-
thyroid hormone related protein [82]; trimethylamine
N-oxide induced structure formation in glucocorticoid
receptor [83]; heme-induced folding of histidine-rich pro-
tein II [84], and many others
CONCLUSIONS
Based on the data summarized above, a typical natively
unfolded protein is characterized by: (a) a specific amino-
acid sequence with low overall hydrophobicity and high net
charge; (b) hydrodynamic properties typical of a random
coil in poor solvent, or PMG conformation; (c) low level of
ordered secondary structure; (d) the absence of a tightly
packed core; (e) high conformational flexibility; (f) its ability
to adopt relatively rigid conformation in the presence of
natural ligands; and (g) a ‘turn out’ response to environ-
mental changes, with the structural complexity increase at
high temperature or at extreme pH
ACKNOWLEDGEMENTS
Iam grateful to Dr P Souillac for the careful reading and editing of the
manuscript This work was supported in part by fellowships from the
Parkinson’s Institute and the National Parkinson’s Foundation
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