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Keeping it in the family: folding studies of related proteins

Kannan Gunasekaran*†, Stephen J Eyles‡, Arnold T Hagler*†§
and Lila M Gierasch*†
Investigators have recently turned to studies of protein families Several recent experimental and computational studies
to shed light on the mechanism of protein folding. In small have sought to elucidate the relationship between folding
proteins for which detailed analysis has been performed, mechanisms of related proteins and the conservation of
recent studies show that transition-state structure is generally sequence information. The variable nature of the available
conserved. The number and structures of populated folding experimental data strongly influences the questions that
intermediates have been found to vary in homologous families can be posed. Specifically, small proteins (<100 residues)
of larger (greater than 100-residue) proteins, reflecting a usually fold by two-state kinetics [5]. Detailed kinetic stud-
balance of local and global interactions. ies have compared the folding/unfolding rates of related
proteins and their transition-state structures. Two parame-
Addresses ters frequently used to characterize the transition-state
Departments of *Biochemistry and Molecular Biology, † Chemistry, and structure are: βT, the relative change in solvent accessibil-
‡ Polymer Science and Engineering, University of Massachusetts,
ity achieved in the transition state (‡), with respect to the
Amherst, MA 01003, USA
§ Structural Proteomics, 9640 Towne Center Drive, San Diego, total change from unfolded (U) to native (N) states
CA 92121, USA [βT = m‡–U/(mN–‡–m‡–U), where the m values are slopes of
plots of the log of the rate constant (lnk) versus denaturant
Correspondence: Lila M Gierasch; concentration] [5]; and φ, which is the change in the free
energy of activation upon mutation of a given residue over
Current Opinion in Structural Biology 2001, 11:83–93 the total change in equilibrium stability for the mutant pro-
0959-440X/01/$ — see front matter
tein (φ = ∆∆G‡–U/∆∆GN–U) [6]. The φ value gives a measure
© 2001 Elsevier Science Ltd. All rights reserved. of the participation of a given residue in the transition state;
φ of 0 indicates that the particular residue does not partici-
pate in a well-formed interaction and φ of 1 that it does. In
ACBP acyl coenzyme A-binding protein
AcP acylphosphatase studies of larger (>100 residue) and multidomain proteins,
ADA2h activation domain of procarboxypeptidase A2 comparisons of related proteins have focused on whether
apoMb apomyoglobin intermediate states are kinetically populated, the nature of
CD2.d1 domain 1 of immunoglobulin CD2 these states and the rates of folding/unfolding as indicators
CoC conservatism of conservatism
CRABP cellular retinoic acid-binding protein of conservation of folding pathways.
Csp cold shock protein
fn fibronectin-like domain Has evolution selected for folding mechanism?
GuHCl guanidinium hydrochloride Computational approaches
IFABP intestinal fatty acid-binding protein
Ig immunoglobulin Recent work by Ptitsyn and Shakhnovich has exploited
iLBP intracellular lipid-binding protein the availability of large numbers of sequences to extract
ILBP ileal lipid-binding protein folding information from evolutionarily related proteins.
Lb leghemoglobin Ptitsyn and Ting [7•] looked for conserved residues among
PDB Protein Data Bank
SH Src homology
globins. This approach led to the identification of two con-
TN tenascin served clusters, one clearly involved in interactions with
the heme and the other, a group of large nonpolar residues
on helices A, G and H. The authors suggested that inter-
Introduction actions among these residues could be critical to initiation
Structurally homologous proteins have embedded in their of folding in the globin family, pointing out that indeed the
sequences the requisite blueprints for their common fold. A, G and H helices form early in apomyoglobin (apoMb)
Can one then assume that the mechanisms of folding for folding [8]. Interestingly, this prediction has been called
structurally homologous proteins will be the same? In into question in a recent experimental study comparing
related proteins that are evolved from a common ancestor, the folding of leghemoglobin (Lb) with that of myoglobin
are folding nucleation sites or transition states conserved? [9•], as discussed below.
Are the roles of particular residues or pairs of residues in
folding conserved? The arrival of the ‘postgenomic era’ In order to identify amino acids conserved for folding and
heralds a time of abundant information from protein eliminate those conserved for function, Mirny and
sequences [1], greatly increased numbers of solved protein Shakhnovich [10•] developed the ‘conservatism of conser-
structures [2,3] and well-developed methodology for both vatism’ (CoC) approach, in which they compare members of
computational and experimental studies of protein folding superfamilies, that is, proteins that share folds, but are highly
[4], positioning investigators to address these questions. divergent in sequence and function. Not surprisingly, sites
84 Folding and binding

Figure 1

Immunity protein Im7 Acyl coenzyme A-binding Apomyoglobin src SH3 domain Cold shock protein

Immunoglobulin TI 127 Intestinal fatty acid-binding Protein G Acylphosphatase Hen egg-white lysozyme

Current Opinion in Structural Biology

Structures of examples from each of the families discussed in this review. Drawings were prepared with MOLSCRIPT [58] and Raster3D [59] from
files in the PDB [3].

of very high CoC are strongly correlated with low solvent contiguous regions, which favor transient formation of
accessibility and, consequently, are generally important in metastable units of secondary structure, and on helix–helix
stabilizing the native state. Additional positions where the packing, which leads to productive folding upon their col-
CoC was much higher than expected from solvent accessi- lision with the appropriate interaction partners. Recent
bility coincided with positions with high φ values in comparisons of families of helical proteins illustrate the
domain 1 of immunoglobulin CD2 (CD2.d1) [11•], CheY impact of these two factors on folding mechanisms.
[12] and the activation domain of procarboxypeptidase A2
(ADA2h) [13], indicating a role in their transition states. Radford and co-workers [18••] carried out a detailed com-
parison of the kinetics and thermodynamics of folding of
Provocatively, using sequence entropy as the measure of two small bacterial immunity proteins, Im7 and Im9,
conservation, Baker and co-workers [14•] drew a different which are 60% identical in sequence (Table 1) and form
conclusion. They found that only one of the six proteins essentially identical four-helix bundle structures
previously characterized by protein engineering and (Figure 1). Intriguingly, both the native state stabilities
kinetic analysis showed any correlation between high and kinetic mechanisms of folding are different. The
φ values and high sequence conservation. These authors more stable Im9 shows two-state folding throughout a
concluded that evolutionary pressures do not act by fine wide range of conditions, even in the presence of sodium
control of sequence to insure formation of a productive sulfate, which is known to stabilize intermediate states.
folding nucleus in the transition state, but instead that the Im7, by contrast, folds via a compact intermediate state.
structural locations of residues with high φ values are As pointed out by the authors, the higher helix propen-
conserved in related sequences, in support of their hypoth- sity of helix I in Im7 compared with Im9 may lead to the
esis that topology governs folding rates and the nature of populated intermediate.
transition states [15,16,17•].
The role of conserved residues in folding has been
Folding of structurally related helical proteins: explored in a recent study of the helical two-state folding
a balance of helix propensities and interhelix acyl coenzyme A-binding protein (ACBP). Poulsen and
docking co-workers [19] followed up their earlier comparison of
The folding of predominantly α-helical proteins is gener- homologous bovine, rat and yeast ACBPs (Table 1) by car-
ally agreed to rely strongly on the helical propensities of rying out extensive mutagenesis and φ value kinetic
Folding studies of related proteins Gunasekaran et al. 85

Table 1

Folding parameters for selected structurally related proteins of up to 100 residues.

Protein family Structural Length ∆G0UN k0f k0u βT* Contact Sequence References
class (number of (kcal/mol) (s–1) (s–1) order conservation
amino acids) (%) (%)‡

Immunity proteins
Im7 α 87 4.0 Burst, 345 1.43 0.92 10.6† – [18••]
Im9 α 86 6.24 1450 0.0124 0.94 12.1† 60 [18••]
Acyl coenzyme A-binding proteins
Bovine α 86 8.19 4105 1.5×10–3 0.57 14.0† – [19,20]
Rat α 86 6.08 395 6.7×10–3 0.69 NA 84 [19]
Yeast α 86 8.22 279 1×10–4 0.61 NA 51 [19]
Cold shock proteins
B. caldolyticus β 67 4.8 1370 0.64 0.93 NA 82 [30]
B. subtilis β 66 2.7 689 9.93 0.91 15.9† – [30]
T. maritima β 67 6.26 565 0.018 0.86 NA 63 [30]
Ig-like domains
FNfn10 β 94 9.4 240 2.3×10–4 0.5 17.0 4 [36••]
TI 127 β 89 7.5 32 4.9×10–4 0.9 17.7 – [36••]
CD2.d1 β 98 6.8 18 1.7×10–3 0.7 17.5 10 [36••]
TNfn3 β 90 5.3 2.9 4.6×10–4 0.7 17.1 8 [36••]
TWIg18′ β 93 4.0 1.5 2.8×10–4 0.7 19.7 9 [36••]
FNfn9 β 90 ~1 0.3 5×10–2 0.7 18.3 12 [60]
SH3 domains
Fyn β 59 6.0 94 9.9×10–4 0.68 18.3 38 [25]
Src β 62 4.1 56.7 0.1 0.69 19.8† – [27]
α-Spectrin β 62 3.8 8.5 0.06 0.69 19.1† 27 [24]
PI3 kinase β 84 3.4 0.35 6.7×10–4 0.60 16.7† 19 [26]
Ig-binding proteins
Protein G β 57 6.6 412 0.1 0.8 17.6 – [44•]
Protein L β 56 4.6 60.6 0.02 0.75 17.2† 15 [43•]
Acylphosphatase family
ADA2h αβ 81 4.1 900 0.65 0.73 17.0 13 [61]
U1A αβ 102 9.3 316 6.3×10–5 0.74 16.9 11 [62]
HPr αβ 85 4.5 14.9 2.1×10–3 0.64 18.4 9 [63]
MerP αβ 72 3.6 1.2 2.6§ NR 18.9 16 [64]
AcP αβ 98 5.1 0.24 6.5×10–5 0.77 21.2 – [46••]

*βT = m‡–U/(mN–‡–m‡–U), where the m values are slopes of plots contact_order [37].
of lnk versus denaturant concentration. †If not given in the literature ‡Sequence conservation is noted relative to the member of the family
reference, contact order was calculated from structures available marked with a dash. §Unfolding rate for 3 M GuHCl final conditions.
in the PDB [3] using the program available at NA, no structure coordinates available; NR, not reported.

analysis of the bovine protein [20]. The authors explored apoMb, suggests common features in the folding pathways
the roles of eight hydrophobic residues that are highly of these two proteins, but clear differences in the nature of
conserved in ACBP sequences and concluded that they the intermediate states [9•] (Table 2). Both proteins popu-
play a critical role in the transition state by forming three late a kinetic intermediate within the burst phase of
mini-cores and mediating zipper-type specific interac- stopped-flow folding kinetics. However, Wright and
tions between helices [20]. Interestingly, earlier work co-workers [9•] showed that helices G and H interact with
showed that yeast ACBP folded and unfolded substan- helix E in the kinetic intermediate for Lb folding. In con-
tially faster than rat or bovine ACBP, with a significantly trast, in the apoMb kinetic intermediate, helices G and H
lower denaturant dependence of its folding rate [19] interact with helix A and, to a lesser extent, with helix B
(Table 1), suggesting the occurrence of a structurally [8]. The authors attribute the changes in the folding inter-
distinct, lower energy transition state. Six polar to mediate to alterations in the sequences of the helices that
hydrophobic substitutions with respect to the rat and form interfaces for interhelical packing, although they
bovine proteins may stabilize helix–helix interactions and point out that altered helical propensity could also change
thus accelerate folding. the extent to which a given helix participates in the fold-
ing intermediate. Consistent with Ptitsyn and Ting’s [7•]
Careful analysis of the folding pathway of a larger helical prediction from analysis of sequence conservation, residue
protein, soybean Lb, a monomeric hemoglobin that is interactions involving helices G and H are critical to both
believed to share a common ancestor with sperm whale apoMb and Lb folding.
86 Folding and binding

Table 2

Folding parameters for selected structurally related proteins of greater than 100 residues, with multiphasic folding kinetics and
structural characterization of intermediates.

Protein family Structural Length ∆G0UN kf Sequence Nature of References

class (number of (kcal/mol) (s–1) conservation** intermediate(s)
amino acids) (%)

apoMb α 151 4.1 3.1, 1.88* – αA,[B],G,H formed [65]
Lb α 143 NR 3.1, 0.93† 13 αE,G,H formed [9•]
Intracellular lipid-binding proteins
CRBP II β 133 8.2 Burst, 2089, 7‡ – Lacks secondary structure [42]
IFABP β 131 5.4 Burst, 31.1‡ 29 Lacks secondary structure [41•]
ILBP β 127 4.5 3.38, 0.59‡ 26 Molten globule [41•]
CRABP I β 136 6.2 Burst, 13.8, 0.8‡ 34 At least two intermediates [42]
Burst, 10, 0.83§ First collapsed with [66,67]
secondary structure, then
β-molten globule
c-type Lysozymes
Hen egg-white αβ 129 NR Burst, 15, 5# – α-Domain formed [47]
Human αβ 130 NR Burst, 300, 15, 5# 59 αA,B, then α-domain [48]
Equine αβ 129 Non-two-state Burst, 333, 4.9# 48 αABD, then heterogeneous [50]
Bovine α-lactalbumin αβ 123 Non-two-state Burst, 0.022¶ 38 α-Domain intermediate [51]
Burst, 20.2¥

*Rate determined by pH jump to pH 5.73. †Refolded into 0.75 M (10 mM EDTA), pH 7.2. ¥Refolding in the presence of 10 mM Ca2+,
urea, pH 6.0. ‡Extrapolated to 0 M denaturant (IJ Ropson, personal pH 7.2. **Sequence conservation is noted relative to the member of
communication). §Refolded into 0.54 M urea, pH 8.0. #Folded into the family marked with a dash. CRBP, cellular retinol-binding protein.
0.54 M GuHCl, pH 4.5. ¶Refolding in the absence of Ca2+ NR, not reported.

Taken together, these studies emphasize that local struc- domains participate in non-native interactions that were
tural propensity, along with helix packing interactions, evolutionarily selected to stabilize the transition state and
govern the structure and stability of the intermediate and thus accelerate folding [28]. As an example, these authors
transition states for related helical proteins. pointed to L33 in α-spectrin SH3 and the corresponding
I34 in Src SH3, both of which have abnormal φ values
Folding of structurally related β-sheet (<0 or >1) [17•,21,23,29].
proteins: topology, conserved cores and native
state stability Prokaryotic cold shock proteins (Csps) comprise another
The small size and simple topology of SH3 domains family of closely related small β proteins [30,31•,32]. These
(Figure 1) has led to several detailed folding studies five-stranded β-barrel proteins fold rapidly, with no inter-
[17•,21–27]. These two-state folders are made up of three mediate states populated. A comparison of Csps from a
β hairpins arranged to form an orthogonal β-sandwich mesophile, thermophile and hyperthermophile had shown
structure. A protein engineering kinetic analysis of the Src that differences in stability arose from altered unfolding
domain by Baker and colleagues [21] yielded a detailed rates (Table 1) [30]. Folding rates were therefore strikingly
picture of the transition state, in which the distal loop hair- similar, despite numerous sequence variations. The transi-
pin and a short 310 helical turn are highly structured in the tion state was very native-like for all three proteins. The
ensemble. In parallel with this work, Martinez and Serrano homologous Bacillus subtilis [31•] and Escherichia coli [32]
[17•] described the transition state of the α-spectrin SH3 Csps also have in common the two-state nature of their
domain by measuring φ values at points equivalent in the folding reaction and a native-like transition state (high βT),
structure to those studied in Src. A remarkable similarity as well as rapid folding and unfolding.
was observed between the two proteins, although the
sequence identity is only 27% (see also Update). We note Ig-like β-sandwich domains have provided a fertile area
that kinetic analysis of the Fyn [25] and PI3 kinase [26] for comparative folding studies. These domains are just
SH3 domains (Table 1) reveals a correlation of folding rate under 100 amino acids in length and some examples fold
with native state stability (as pointed out by Clarke and by two-state kinetics, whereas others have populated
co-workers for the Ig-like domains, discussed below). intermediates. The Ig-like structure is one of the most
common folds and is adopted by several different super-
A recent theoretical study using lattice simulations families of diverse function and unrelated sequence
hypothesized that some conserved residues in SH3 [33,34]. Parker et al. [35] explored the nature of the folding
Folding studies of related proteins Gunasekaran et al. 87

intermediate in CD2.d1, which folds by a three-state path- Figure 2

way. They identified a set of highly conserved residues in
the core BCEF strands (Figure 2) that were the most TWIg18′ and TI 127
structured in the intermediate. More recently, this group
showed that this folding intermediate was stabilized by
core mutations with higher β-structure propensity, which A
in turn destabilized the native structure, thus arguing that D E B G F C
the β-strand conformation of the polypeptide chain is
A′ C′
established in the intermediate [11•]. φ value analysis and
double-mutant studies showed that three residues in the
BCEF core β structure (Figure 2) form a tightly packed
hydrophobic nucleus [11•].

Clarke et al. [36••] compared the folding of five proteins with

widely varying functions, representing two different Ig-like CD2.d1
superfamilies, to distinguish residues conserved for folding
and residues conserved for function (the ‘fold approach’).
These proteins consist of two fibronectin type III domains D E B A G F C C′ C′′
and three Ig-like domains, which share topology (Figure 2)
but no discernible sequence homology. Combining their
data with published data for a sixth Ig-like domain extended
the range of folding rates to almost three orders of magni-
tude (Table 1). Intriguingly, the folding rates were found to
vary directly with protein stability. The authors noted that
previous surveys of protein folding had not found this corre-
lation [5,37]. These six proteins have similar contact orders
TNfn3 and FNfn10
by virtue of their common structure, bringing into question
Baker’s simplifying correlation [15,37]. The authors thus
hypothesized that several residues in the common core of
this fold, namely the BCEF and G strands (Figure 2), con- E B A G F C C′
stitute a folding nucleus in this class of proteins, consistent
with the earlier work discussed above [11•,35].

In a subsequent study, Clarke and co-workers [38•] tested

Current Opinion in Structural Biology
their hypothesis using a detailed protein engineering
analysis of the third fibronectin type III domain of human
Topology of representative Ig-like proteins, showing strands B, C, E, F
tenascin (TNfn3). They concluded that a ring of four and G, which form a common core in the transition state for this family.
residues in the BCEF core, including the three residues
identified in CD2.d1 [11•], is partially structured in the
transition state. Interestingly, the CD2.d1 and tenascin acids in length and fold by complex mechanisms with pop-
φ value data appear to be in conflict in some regions; low ulated intermediates. Dalessio and Ropson [41•] concluded
φ values had been reported for the CE insert in CD2.d1 that intestinal fatty acid-binding protein (IFABP) and ileal
[11•], in contrast to the intermediate φ values displayed by lipid-binding protein (ILBP), which are 23% identical, fold
the corresponding C′ strand of TNfn3. Hamill et al. [38•] through different pathways (Table 2). In the case of ILBP,
argued that the difference arises from the different lengths the authors conclude that a compact molten globule-like
of the loops and that shorter length leads to an obligate kinetic intermediate with substantial secondary structure
packing of the C′ strand in tenascin when the core strands was populated in both its unfolding and refolding path-
are folded. A similar length-dependent involvement of EF ways. IFABP, on the other hand, appeared to fold via an
loops in the transition state was supported by a comparative intermediate with little secondary structure. These results,
study of four Ig-like domains [39]. together with those of their earlier study [42], led the
authors to propose that there are two productive pathways
The intracellular lipid-binding proteins (iLBPs) are a fam- to the formation of the iLBP fold and that features of the
ily of even larger β proteins whose folding is being probed. hydrophobic core may govern which of these is followed.
They are structurally well conserved, consisting of two
five-stranded antiparallel orthogonal β sheets wrapped In recent sequence analysis, we have identified a network
around a central ligand-binding cavity (solvent-filled in the of conserved long-range interactions in cellular retinoic
apo-proteins), despite wide variations in sequence conser- acid-binding protein (CRABP), a member of the iLBP fam-
vation [40]. These proteins are approximately 130 amino ily (K Gunasekaran, AT Hagler, LM Gierasch, unpublished
88 Folding and binding

Figure 3




Current Opinion in Structural Biology

data). The network includes a hydrophobic and a polar clus- protein L (Table 1, Figure 1). These small proteins share a
ter; in some family members, the polar cluster is another strikingly similar topology, consisting of a β hairpin, an
hydrophobic cluster instead. These sequence variations are α helix and a second hairpin, but their folding is distinctly
clearly consistent with common structures, but may lead to different. The first β turn of protein L is significantly
divergent folding pathways. formed in the folding transition state, whereas the remain-
der of the structure does not condense until after the
Overall, the folding of β-sheet proteins appears to be gov- rate-limiting step, possibly because of the strain involved in
erned by nonlocal interactions in the transition state, not forming the second turn (three consecutive φ angles are
unexpectedly, given the nature of β-secondary structure. positive) [43•]. In contrast, the structurally similar, but
In addition, the transition states are generally highly struc- sequence-divergent, protein G (identity to protein L is only
tured. In two cases, folding rates are convincingly 15%) folds with only the second β turn formed in the tran-
correlated with native state stability, arguing that interac- sition state [44•]. The authors attribute these results to the
tions that stabilize the native state are present in the presence of more favorable contacts in the second turn of
transition state. protein G. Indeed, a peptide derived from this region of the
protein G sequence forms a native-like turn structure in
Mixed α/ββ protein families: a mixture of aqueous solution [45]. Taken together, these observations
folding behaviors suggest that there may be significant differences in transi-
Families of proteins with a mixture of α helices and tion states among structurally related α/β proteins and that
β sheets display a wide range of comparative folding para- the origin of this may be variations in local sequence
meters. The simplest α/β examples comprise the propensities. In this way, these results are reminiscent of
Ig-binding domains of streptococcal protein G and the findings for α-helical protein family members.
Folding studies of related proteins Gunasekaran et al. 89

Figure 3 legend

We may shed light on factors affecting the folding trajectories of larger loops. In this case, the structured portion of the transition state
related proteins that fold by two-state mechanisms and propose (enclosed within the dashed box) contains elements of the four
explanations for seemingly inconsistent experimental results by going secondary structures packed together via identical interactions in the
back to basic definitions. Through classical transition-state theory, the Hφ , ∆Hstrain and
two transition states. To a first approximation, ∆G‡–U ‡–U
folding and unfolding rates are determined by the free energies of ∆HNB‡–U
will be the same for both proteins, as the substructures in the
activation from either the unfolded or the native state, respectively: transition state are identical, and the remainder of the chain is the
same in the unfolded and transition states. Therefore, the only
k f = Cexp[ − ∆G‡ −U /RT]; k u = Cexp[ − ∆G‡ −N/RT] difference between the two chains will be the chain entropy. The
longer loops in the second protein result in a higher entropy change
Here, ∆G‡–U and ∆G‡–N are the heights of the transition state relative and consequently a larger ∆G‡–U and a slower folding rate. This
to the unfolded or native structures, respectively, and C is the pre- entropy effect is exactly what contact order measures and thus, in
exponential constant. Focusing on the folding direction, we can write these cases, one would find that the folding rate is related to the
∆G‡–U in terms of its energetic and entropic contributions: contact order, as proposed by Baker and colleagues [15,16,17•,37]
and, in fact, quantitated by Fersht [68•]. Among the protein families
∆G‡–U = ∆H‡–U − T∆S‡–U
described in this review, the best example of the pre-eminence of this
The enthalpy and entropy can be further partitioned: entropic term is the AcP family (Table 1) [46••]. (We caution that it is
really the contact order of the transition state, not the native structure,
∆H‡ − U = ∆HH‡ −φU + ∆Hstrain
‡ −U + ∆H‡ −U + ∆H‡ −U + ∆H‡ −U
elec VDW HB
that determines the folding rate in this case. Only to the extent that the
contact order in the transition state mirrors that of the native or the
∆S‡–U = ∆SH‡ −φU + ∆Sstrain
‡ −U + ∆S‡ −U + ∆S ‡ −U + ∆S ‡ −U + ∆S ‡ − U
elec VDW HB chain
topology of transition states in the proteins being compared is the
same will a correlation of folding rate and contact order be obtained.)
where ∆H‡–UHφ
is the difference in the hydrophobic enthalpy between the
transition state and the unfolded state; ∆Hstrain
‡–U is the difference in In the next limiting case, depicted in (b), the proteins have identical
strain energy in the bonds, bond angles and torsion angles of topologies and transition-state substructures, which are also the same
the protein between the two states; ∆Helec ‡–U , ∆H‡–U and ∆H‡–U are the
as in the native structures. In this case, however, interacting residues in
corresponding electrostatic, van der Waals and hydrogen-bonding the transition-state substructures differ because of specific sequence
contributions, respectively. The same terms occur in the difference in changes (indicated by empty versus filled circles). Thus, ∆GHφ ‡–U
entropy between the two states, with one additional term, viz., the and ∆HNB ‡–U will differ because the residue–residue interactions differ,
contribution to the difference in the chain entropy between the but the chain entropy will be identical in the two cases. Hence, the
unfolded and transition states, ∆Schain
‡–U . folding rate will be directly related to the stability of the structural
element in the transition state. As in this limiting case the transition-
The difference in electrostatic, van der Waals and hydrogen-bonding state substructures are native-like, the difference in stability of the
energy can be combined into one contribution, ∆H‡–U NB
, the transition-state structure will mirror the differences in stability of the
intramolecular nonbonded energy of the chain (for our purposes here, native structure. The folding rate will not be related to contact order,
interactions with solvent are included in the hydrophobic component). but instead to the stability of the native structure. This latter situation is
The difference in electrostatic, van der Waals and hydrogen-bonding reflected in the results that Clarke et al. [36••] obtained in the case of
entropy is in general small and can be neglected. The valence (or the Ig-like proteins (Table 1). An interesting variant on this second
strain) entropy is basically reflective of differences in vibrational limiting case is presented by the Csps from three different strains of
frequencies and is also small and can be neglected. So, these bacteria that grow at different temperatures [30]. Here, the native
equations simplify to: states vary in free energy with respect to the unfolded state, but
folding rates differ by less than threefold. Instead, the differences in
∆H‡–U = ∆HH‡ −φU + ∆Hstrain
‡ −U + ∆H‡ − U
native state stability are reflected primarily in differences in the
∆S ‡–U = ∆SH‡ −φU + ∆Schain
unfolding rate, which are nearly three orders of magnitude. One is
‡ −U
forced to conclude that interactions that differentially stabilize the
Grouping the hydrophobic contributions, we can write the free energy, native state are not present in the transition state (schematically
∆G‡–U, as: indicated by arrows in Figure 3b).
∆G‡–U ≈ ∆GH‡ −φU + ∆Hstrain
‡ − U + ∆ H ‡ − U − T∆ S ‡ − U
NB chain
The analysis of these limiting cases provides an explanation for the
It is instructive to examine some limiting examples to hone our apparently contradictory results for the AcP, the Ig-like and the Csp
expectations. families. Additionally, we are able to develop working models for the
nature of the transition states for these protein families from this
In the first limiting case, depicted schematically in (a), two proteins reasoning. Of course, these models are ‘simplified’ and real
(I and II) have identical sequences except for insertions in the second cases are not likely to be amenable to such separation of free
protein relative to the first (shown as bold lines), resulting in much energy components.

Another family of structurally similar small α/β proteins argue that this is a result of a very different relative con-
studied recently includes acylphosphatase (AcP) and tact order in the two proteins. In fact, a strong correlation
ADA2h (Table 1, Figure 1). Although these proteins are was seen between relative contact order and the lnkf for a
not closely homologous, they share a similar overall topol- number of other proteins with the same topology (HPr,
ogy (βαββαβ). Similarities have been observed between MerP and U1A) (Table 1) (see the review by Oliveberg in
the transition-state structures of these two proteins, this issue, pp 94–100).
despite a mere 13% sequence identity [46••]. Although
their folding proceeds through a similarly structured Folding studies of the larger α/β c-type lysozymes and the
native-like transition state (high βT), their folding rates homologous α-lactalbumins have revealed differences in the
differ by three orders of magnitude (Table 1). The authors nature of intermediate states, although they have similar
90 Folding and binding

kinetics. For example, in hen egg-white lysozyme, the correlation of unfolding rate with native state stability,
entire α-helical domain folds before docking to the more whereas folding rates in the AcP family correlate well with
slowly folding β-sheet domain to form the final folded struc- contact order. Can one reconcile these results? As folding
ture [47]. On the other hand, human lysozyme initially rate variations arise directly from changes in the height of
forms a structure involving only the first two helices on a the transition-state barrier for two-state folders, one must
faster time-scale than complete α-domain formation [48]. explore how sequence variations could affect the free ener-
Side-by-side comparisons of folding rates using mass spec- gy of the transition state. We have analyzed simple limiting
trometry revealed subtle differences in rate constants in cases to explore how the nature of the transition states
other naturally occurring variants [49]. The calcium-binding could lead to different rate trends (see Figure 3). We con-
equine variant forms an early intermediate with three of the clude that the apparently contradictory folding behaviors
four helices folded, followed by heterogeneous assembly to arise from situations in which different terms such as chain
the native state [50]. Bovine α-lactalbumin folds via an entropy and nonbond energy dominate in the free energy
intermediate state with similarities to the equilibrium par- of activation for folding.
tially folded A-state structure, although with many fewer
persistent contacts than observed for the kinetic intermedi- A critical question that has not been treated in any of the
ate in lysozyme [51]. Recently, chimeric proteins with recent work on the folding of related proteins is what
elements of sequence from various lysozymes and α-lactal- characteristics of folding mechanisms are subject to evo-
bumins were found to fold in a somewhat additive manner, lutionary pressures. Some computational papers have
with the individual elements reminiscent of their parent suggested that evolution selects for rapid protein folding
protein [52]. These data lend strong support to the idea that [53,54]. Yet the biological requirements on most proteins
local sequence drives structural formation and that the pro- are certainly more complex than these simple ideas. Some
teins complete the folding process via an evolutionarily proteins may need to fold quickly and efficiently to per-
conserved transition-state structure. form their cellular task; an example may be ubiquitin. In
other cases, the rate must be slow enough to allow them
Conclusions to be correctly recognized by molecular chaperones for
The past two years have seen considerable research activ- subsequent localization to organelles, assembly into com-
ity harnessing protein families to address the protein plexes and/or post-translational modification. Still others
folding problem. As shown in this review, even with com- may be selected to avoid unfolding by kinetically trap-
parative behaviors of family members in hand, in-depth ping the native state, as in the case of α-lytic protease
understanding is elusive because it requires knowledge of [55]. Unexpectedly, recent work suggests that folding in
the energetics and structures of states along a folding the cell may be purposefully inefficient, so that a sub-
trajectory that we do not have at this time. Nonetheless, stantial fraction of translation products do not fold
this survey of a number of different protein families makes successfully, are therefore degraded and then are present-
several points clear. (1) Transition-state structures are fre- ed on the surface of the cell as indicators of the synthetic
quently conserved in structurally homologous proteins. activity of the given cell to help warn the immune system
Table 1 shows that the βT values rarely vary much within a when foreign proteins are being made [56,57]. Taken
family and φ value analysis of the SH3 domains, the AcP together, these examples remind us that we must remain
family and the Ig-like domains shows preservation of the cognizant of the evolutionary pressures for successful
residue positions that are in well-formed interactions in the in vivo folding.
transition state. Similarly, the finding that residues with
high φ values were among the most conserved in the ACBP Update
family supports this idea. Characterization of intermedi- A recent study from Guerois and Serrano [69] questions
ates for the lysozyme family also argued that the proposition that structurally related proteins fold by
transition-state structures are conserved. (2) Exceptions to common mechanisms. These authors report the results of
common transition state behavior are the immunity pro- a protein engineering analysis of the folding kinetics of
teins, the globins and proteins G and L. In each of these Sso7d, a DNA-binding protein from the hyperthermophile
cases, authors attributed the different folding pathways to Sulfolobus solfataricus [70]. Although there are some dis-
differences in local sequence propensities. We conclude tinct differences between the structure of Sso7d and that
that it is possible to achieve the same fold by varying com- of the previously studied SH3 domains, such as a C-termi-
binations of local and global sequence influences. It is nal α helix in place of a β strand that is part of a
likely that this is the origin of the observed differences in three-stranded β sheet in the SH3 domains, these authors
folding mechanisms for the iLBP family, but the available argue that Sso7d has an SH3-like fold. They draw the con-
data do not yet say. (3) Even in families for which the tran- clusion from their φ value analysis that a different
sition-state structure appears to be conserved, folding rates hydrophobic core forms in the transition state of Sso7d
vary widely. Folding rates in the Ig-like family correlate than in either the Src or α-spectrin SH3 domains. Their
with native-state stability, as pointed out by Clarke and co- conclusion, which they justify using a new folding predic-
workers, and a similar trend emerges when data for the tion program, Fold-X, is that topology alone is insufficient
SH3 domains are compared (Table 1). The Csps show a to determine the nature of the transition state and that
Folding studies of related proteins Gunasekaran et al. 91

sequence features must be invoked to account for the its folding pathway, with helices G and H and part of helix E formed. This
result contrasts with the early formation of helices A and B, along with
observed folding mechanism. helices G and H, in apoMb.
10. Mirny LA, Shakhnovich EI: Universally conserved positions in
A neural network approach has been exploited by Dinner • protein folds: reading evolutionary signals about stability, folding
and Karplus [71] to explore the relationship between fold- kinetics and function. J Mol Biol 1999, 291:177-196.
The authors developed an approach using structure-based sequence
ing rates and both contact order and native-state stability. alignment of different proteins belonging to families that have distinct func-
These authors point out that their calculations show that tions in order to eliminate the conservation of residues involved in the func-
tion and focus on those that are conserved for folding. The authors
proteins with small contact orders, which are predominant- conclude that positions identified by this ‘conservatism of conservatism’
ly α-helical, always fold rapidly, whereas proteins with (CoC) approach correlate with the residues that have high φ values in pro-
tein engineering studies. Several examples are discussed in this paper, but
large contact orders, which may be α/β or predominantly β, φ values are not available for many of the residues that are shown to have
fold with rates varying over three orders of magnitude. high CoC.
Clearly, in these latter proteins, factors other than contact 11. Lorch M, Mason JM, Clarke AR, Parker MJ: Effects of core mutations
order affect the rates of folding. They were able to train • on the folding of a β-sheet protein: implications for backbone
organization in the I-state. Biochemistry 1999, 38:1377-1385.
their neural network to predict these highly varying rates The authors examined the impact of several mutations introduced into
of α/β and β-sheet proteins, and to show that native state β strands in the core of CD2.d1. Six out of nine mutations that stabilize the
native state also stabilize the major intermediate along the folding pathway
stability played a significant role in determining folding of this immunoglobulin domain. This result demonstrates that the β confor-
rates for proteins in these structural classes. mation of this region is established in the intermediate. Extensive φ value
analysis and double-mutant studies were also carried out to characterize the
folding of this protein.
Acknowledgements 12. Lopez-Hernandez E, Serrano L: Structure of the transition state of
We thank Ira Ropson for communicating unpublished results. We wish to folding of the 129 aa protein CheY resembles that of a smaller
express our gratitude to the members of the Gierasch laboratory for critical protein, CI2. Fold Des 1996, 1:43-55.
reading of the manuscript. The preparation of this review was supported in
part by National Institutes of Health grant GM27616 to LMG. 13. Villegas V, Martinez J, Aviles F, Serrano L: Structure of the transition
state in the folding process of human procarboxypeptidase A2
activation domain. J Mol Biol 1998, 283:1027-1036.
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