Báo cáo khoa học: The importance of being dimeric pptx

Other important features such as surface hydrophobicity, internal empty and⁄ or water filled cav-ities, hydropathic distribution of amino acid residues have often been found to play a sig

The importance of being dimeric

Giampiero Mei1,2, Almerinda Di Venere1,2, Nicola Rosato1,2and Alessandro Finazzi-Agro`1

1 Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Rome, Italy

2 INFM, University of Rome ‘Tor Vergata’, Rome, Italy

Introduction

The world of globular proteins appears, to a naive

observer, to be very complex At first sight it is even

difficult to find any regularities that (may) exist

In particular, the ability of these macromolecules to

reach their final shape among the many different

con-formations in a very short time is astonishing Small,

globular proteins usually show some interesting

corre-lations between their structural features and the

ther-modynamic parameters characterizing their overall

stability [1] Other important features (such as surface

hydrophobicity, internal empty and⁄ or water filled

cav-ities, hydropathic distribution of amino acid residues)

have often been found to play a significant role in the

protein folding process [2,3]; perhaps the most crucial

event for cell life

The situation is even more complex in the case of

oligomeric structures as no obvious rules concerning

their molecular mass, amino acid composition, sequence or tridimensional structure are apparent

An inspection of the list of proteins made by more than one polypeptide chain shows a striking feature, namely that of the surprisingly high number of pro-teins made up of two subunits (Fig 1) This finding is even stranger when one realizes that most of these pro-teins are made up of two identical subunits (Fig 1) Let us therefore discuss the meaning of such a pheno-menon Obviously, the explanation seems far simpler when the subunits of a dimer are different In this case, each subunit could have a different role; for example, one subunit may be catalytic and the other regulatory and this may be the reason for dimer formation Simi-larly, it would be understandable if they bound differ-ent molecules with differdiffer-ent affinities

The situation is more intriguing when one tries to figure out the meaning of proteins made by identical subunits Again, one may think that in the case of

Correspondence

A Finazzi-Agro`, Department of Experimental

Medicine and Biochemical Sciences,

University of Rome ‘Tor Vergata’, Via

Montpellier 1, Rome 00133, Italy

Fax: +39 06 72596468

Tel: +39 06 72596460

E-mail: mei@med.uniroma2.it

Note

This paper is dedicated to the late G Weber

and W.E Blumberg who first stimulated our

attention to the problem.

(Received 14 August 2004, revised 17

September 2004, accepted 21 September

2004)

doi:10.1111/j.1432-1033.2004.04407.x

Why are there so many dimeric proteins and enzymes? While for hetero-dimers a functional explanation seems quite reasonable, the case of homo-dimers is more puzzling The number of homohomo-dimers found in all living organisms is rapidly increasing A thorough inspection of the structural data from the available literature and stability (measured from denatura-tion–renaturation experiments) allows one to suggest that homodimers can

be divided into three main types according to their mass and the presence

of a (relatively) stable monomeric intermediate in the folding–unfolding pathway Among other explanations, we propose that an essential advant-age for a protein being dimeric may be the proper and rapid assembly in the cellular milieu

Abbreviations

IAR, interface amino acid range; SLL, squared loop length.

noncatalytic dimers that bind other molecules, such as

DNA, the protein behaves like a pair of tongs to hold

them in a way appropriate for some other action A

simple explanation still applies when a catalytic dimer

has the active site at the interface between the

sub-units However, most catalytic proteins are composed

of identical subunits each containing an ‘active’ site;

thus it remains to be explained why these proteins are

made up of two polypeptide chains instead of being

simply a single chain that is twice as large

The possible advantages provided by a homodimeric

structure were first advanced by Monod, Wyman and

Changeux in their classic paper about allosteric

transi-tions in enzymes [4] In this study they emphasized the

fact that isologous associations (i.e the binding of two

identical subunits, involving identical binding domains)

give rise to ‘closed structures’, with an intrinsic

sym-metry and probably an enhanced stability They also

suggested that in vivo, a fast formation of the

oligo-meric structure might avoid a random association of

its subunits with other cellular proteins Experiments

performed by Koshland [5] on the in vitro folding of

mixtures of different oligomers have confirmed this

hypothesis It was concluded, therefore, that due to

evolutionary selection, the interaction at the

intersub-unit binding site is generally highly specific; its

unique-ness being guaranteed by the rapid formation of each

protomer’s tertiary structure (i.e during or

immedi-ately after ribosomal translation) Furthermore, in the

early 1980s, high-pressure techniques allowed new and

more detailed studies on the oligomers In fact, the

mechanical separation of dimeric protein subunits

induces a conformational drift in the protomers’

struc-ture demonstrating how quaternary interactions can

affect the structure of each monomer [6,7]

In this review we shall present some further possible reasons for the potential advantage of dimeric proteins

in living systems; in particular genetic saving, func-tional gain and structural advantage

Genetic saving

What is the optimum size for an enzyme? Obviously the length of each polypeptide is a compromise between two distinct, but equally important, require-ments: stability and the minimum scaffold necessary to build up the active site Evidently, natural evolution must have accomplished these two goals avoiding any redundancy, i.e without wasting materials Genetic saving may apply when an oligomeric protein is com-pared to a monomer of identical size However, the energetic balance of synthesizing a polypeptide chain is only barely accountable for the whole process Besides the energy needed for binding the amino acids to their tRNAs and then to each other when on the polyribo-somes, one should take into account the energy con-sumed by the synthesis and preprocessing of mRNA inside and outside the nucleus, and that needed to keep the regulation machinery running A naive approximation is that to obtain an mRNA twice as long, one should spend twice the amount of energy Therefore the synthesis of a dimer might require signi-ficantly less energy than that of a monomer of the same overall molecular mass This simplistic assump-tion does not take into account that the probability of errors during the replication of a gene and its transla-tion increases in a way more than proportransla-tional to the gene length Therefore, one should consider the addi-tional cost for the cell to keep the whole process under control Another factor in favor of synthesizing dimers instead of larger monomers might be the different time required for ribosomes to walk across shorter mRNAs

Functional gain

By functional gain we mean any improvement in the catalytic action of enzymes on substrate(s) This effi-ciency is governed among others by ‘mechanical’ fac-tors: (a) the encounter between the two molecules that,

in a diffusion-controlled reaction, depends on bimole-cular quenching rate [8]; and (b) the orientation factor, which takes into account the correct lining up between the substrate to be processed and the active site The bimolecular quenching rate is proportional to the concentration of the enzyme and to the effective hydrodynamic radius at which the enzyme–substrate reaction takes place (often approximated to the protein radius, as the enzyme is generally much larger than the

dimers

hetero dimers

homo dimers trimers

heptamers

tetramers

pentamers

hexamers

octamers

Fig 1 Percentage distribution of oligomeric proteins (dimers,

tri-mers, tetratri-mers, pentatri-mers, hexatri-mers, heptamers and

octam-ers) Oligomers represent  15% of all the crystallographic data

present to date (August 2004) in literature ( 4200 from a total

of 27 000 structures).

substrate) The orientation factor depends instead on

the protein size and, assuming a spherical shape, it can

be approximated by the ratio between the active site

surface and the overall enzyme surface These three

parameters, namely concentration, radius and

enzy-matic surface, play opposite roles as a function of

vol-ume and no practical advantage can be envisaged for

dimers with respect to monomeric proteins

A different explanation may call into play the large

structural modifications that occur more easily in

a multidomain enzyme with respect to a rigid,

mono-meric protein This factor may favor the interaction

between a protein and its ligand according to the

‘induced fit’ model of Koshland [9] It is well known

that oligomers may display allosteric behavior [4]

However, while this phenomenon is observed

fre-quently in the case of multisubunit proteins, it appears

to be far less common in dimers

In conclusion, as the above reported factors seem to

be at least of minor importance, one should try to

dis-cover the peculiar features of dimers for a possible

cor-relation between the stability, folding and functional

properties of dimeric proteins

Structural advantage

Conformational stability and folding

intermediates

A comparison between the stabilization energy per

resi-due for some monomeric and dimeric proteins is shown

in Fig 2 The data demonstrate clearly a similar trend

for both types, i.e an exponential decrease, reaching a

constant value above 400 amino acids per subunit Ten

years ago, Neet and Tim [1] found an approximately

linear correlation between the molecular mass and the

stability of 17 dimeric proteins, although they suggested that this correlation could not hold for heavier oligomers We extended the analysis to some 40 other dimeric proteins using the data available in the litera-ture (Table 1), and also taking into account the pres-ence of intermediate species detected by both kinetic and equilibrium unfolding measurements In particular,

we have divided them into three classes according to the following denaturation patterns: class A, N2« 2U; class B, N2« 2I « 2U; class C, N2« I2« 2U where N2represents the native state, I and I2are inter-mediate monomeric or dimeric species, respectively, and U is the fully unfolded protein Although this clas-sification is somehow weak – in several cases the inter-mediates may be stabilized or destabilized by the solvent properties or by introducing ‘ad hoc’ mutations – it might help to find a possible correlation between the structural properties and the stability of dimeric proteins For example, in the case of globular, mono-meric proteins, the presence of partially folded states seems to be correlated strictly to a delicate balance between the mean charge and hydrophobicity [10] As shown in Fig 3, the pattern appears more complex than described previously, as no linear relationship seems to hold between the overall conformational sta-bility and the size of the proteins In particular, all three data sets are characterized by a monotonic increase in stability, up to a threshold value that varies from 150 amino acids per subunit (class A) to  350 amino acids per subunit (class C) Then the stabiliza-tion energy asymptotically drops to lower values ( 12,

15 and 20 kcalÆmol)1for the three groups) This behav-ior is quite reasonable because a stabilization energy greater than this value could generate ‘indestructible’ proteins unsuited for the continuous making and breaking that characterizes living systems

Conformational stability and catalytic activity The free energy of unfolding is the main parameter characterizing these three groups of dimers Further-more, a functional analysis has shown that only 20% of the proteins of class A reported in Fig 3A are enzymes, with 60% and up to 100% in groups

B and C, respectively This observation suggests that some correlation may exist among stability, size and function As a matter of fact, most of the smaller proteins belonging to class A are DNA (or RNA) binding proteins that possibly require a homodimeric structure only because they have a

‘molecular tweezers-like’ function The situation is more complex for the class B and particularly the class C enzymes

0.00

0.05

0.10

0.15

0.20

aa / subunit

Gu

Fig 2 Free energy of unfolding per residue for monomeric (d) and

dimeric (s) proteins as a function of the total number of amino acids.

Table 1 List of dimeric proteins divided into three main classes according to their unfolding pathways.

Protein

Residue number (per subunit)

DG (kcalÆmol)1)

Protein

Class A

Transcription factor LFB1

Class B

Class C

An important general feature of enzymes is known

to be their local structural flexibility [11] It has been argued that the usually very large ratio between the dimension of an enzyme and that of its active site is related to the possibility of finely tuning catalytic activ-ity Changes in protein shape are thus fundamental in exerting biological control in the cell [12] and for oligomeric proteins, in particular, this has been well known since the seminal studies on allosterism [4] However, besides these cooperative mechanisms, it has been proposed recently that the oligomerization process itself might tune the enzymatic function For example, a structural analysis of several glycolytic enzymes has suggested that significant changes in their enzymatic activities do not require large

conformation-al changes [13,14] It seems that in these cases, the formation of intersubunit contacts influences the biological activity by allowing very subtle conforma-tional changes at the active site in such a way that oligomerization can indeed activate the monomeric subunits These findings are consistent with the small (but significant) conformational changes observed in pressure-induced dissociation experiments [6], even though a generalization of this mechanism to all dimeric enzymes is not yet warranted [13,14]

Insights on dimer intersubunit surface The dissociation free energy (DGdiss) of several

dimer-ic proteins considered in this paper was obtained from equilibrium unfolding measurements The DGdiss

values range from 6 to 15 kcalÆmol)1, generally accounting for more than 50% of the total free energy of unfolding This finding is consistent with the widespread idea that the contacts at the surface, hidden between the monomeric subunits, play a fundamental role in the stabilization of oligomeric proteins Taking advantage of the available crystallo-graphic data, we have evaluated the ratio between the dimeric intersubunit interface value and the total accessible surface area of each monomeric subunit This ratio is not constant, the smaller the subunit size, the larger the contribution of the interface In particular, this ratio shows the highest values for very

Table 1 (Continued).

Protein

Residue number (per subunit)

DG (kcalÆmol)1)

Protein

0

10

20

30

0

10

20

30

40

B

A

C

0

20

40

aa/ subunit

Fig 3 Total free energy of stabilization for dimeric proteins that

undergo a simple two-step denaturation process (A) Class A and a

three-step unfolding process with a monomeric (B, class B) or

dimeric (C, class C) intermediate species Filled symbols represent

those proteins characterized by a linear trend of the DG values vs.

their size.

small proteins of class A (Fig 4A, dashed area) The

crystallographic data indicate that these proteins are

characterized by a very high content of secondary

structure, namely between 60% and 90% The group

is composed mostly by DNA-binding proteins (such

as ROP, ARC repressor, TRP repressor) and

pro-teases [such as Simian immunodeficiency virus (SIV) and

HIV] or protease inhibitors (e.g subtilisin inhibitor)

that, despite different tridimensional structures, share

a common functional role for their dimeric interface

(i.e substrate binding) On the other hand, the

all-or-none transition that characterizes the folding process

of these small dimers strongly suggests that the

assembly of their quaternary structure parallels the

formation of a-helices and⁄ or b-structures Thus, a

high number of intersubunit contacts might be

already formed at the earliest steps of the folding

process Instead, larger dimers display a parallel

increase of both dimeric interface and total accessible

surface area (data not shown), resulting in a constant

percentage of residues present at the interface The contribution of the dimeric interface to the total sur-face buried during folding and dimerization can be evaluated using the algorithm proposed by Miller

et al [15] (Fig 4B) Clearly, the surface hidden at the dimeric interface represents a significant function of the buried residues only for small size dimers, while above a threshold of 100 amino acids per subunit, no differences are apparent among the three classes of dimeric proteins

As the roughness of the monomer contact surface can be critical for the dimerization process, we have also checked for the presence of gaps and voids at the interface of the dimers The data demonstrated that the larger the dimer size, the higher the probability of finding empty (or water-filled) spaces created by the mismatching of the two monomeric surfaces (data not shown) Furthermore, for dimers of a given size, those proteins that have a folding intermediate displayed less empty volumes, reflecting a different ‘pairing attitude’

of the monomeric subunits that characterize the A, B and C groups

Hydrophobic interactions in dimers and the role

of the intersubunit surface Hydrophobic interactions are essentially due to the bur-ial of apolar residues in the interior of proteins As the volume-to-surface ratio increases with the size of a glob-ular molecule, one might have expected that the relative number of hydrophobic residues in a protein also increased with the length of the polypeptide chain Early studies on the ‘hydropathic’ character of proteins [16] have instead demonstrated that the mean hydropathy has a fairly constant value that does not depend on the total number of amino acids Furthermore, it has been found recently that a balance exists between the accessi-bility of hydrophobic and hydrophilic surfaces in most

of 500 proteins [17] independently of the protein molecular mass A possible explanation for these find-ings is the formation of water-filled cavities that arise from packing defects [2] In fact, the cavities accommo-dating water molecules are lined by hydrophilic residues [3,18] The dimeric proteins appear to follow the same rules The hydrophobicity at the subunit interface decreases with the polypeptide size (Fig 5), indicating that for large dimers the hydrophobic bonds can be pro-gressively replaced by polar interactions It appears therefore that the dimers are held together by nonpolar interactions in small proteins, but also by salt bridges and other electrostatic interactions within a suitable scaffold of hydrophobic residues in the large ones Given the rather constant ratio between hydrophobic

0.0

0.1

0.2

0.3

0.4

A

0

10

20

30

40

aa/subunit

B

Fig 4 (A) Fractional contribution of the dimeric intersubunit surface

(DIS) with respect to the total accessible surface area for a

mono-mer (i.e DIS⁄ ASA) as a function of the monomono-mer size (class A, h;

B, d and C, m) (B) Fractional contribution to the total buried

sur-face (DIS ⁄ buried surface) The total buried surface upon folding has

been evaluated according to Miller et al [15] Dashed areas indicate

the largest change of the fractional dimeric interface (see text).

and polar residues, the more polar residues are present

at the interface, the less polar buried in the protein core,

thus, reducing the amount of defects and of

protein-entrapped water This may represent an important

fac-tor for the stability of larger dimers

The importance of being dimeric

Taking into account the main structural and functional

features of the three groups of dimers considered so

far, it is tempting to propose a different explanation

for each case, considering the role played by

dimeriza-tion

Structural functionality: a rationale for smaller

dimers

Small dimers almost all belong to class A and C Their

function is essentially the binding of other molecules,

often in a very specific and symmetric way An

obvi-ous example is that of RNAÆDNA binding proteins

(such as ROP) They usually recognize and bind

speci-fic sequences of nucleic acids only in their dimeric

state, immediately loosing this ability if the ‘hinge’, i.e

the dimer contact, is lost This is a clear example of a

molecular switch (the on–off positions corresponding

to the dimer–monomer states) that can regulate

important functions in living organisms

A strict quaternary structure-to-function relationship

is obviously not limited to DNAÆRNA binding

pro-teins For instance, the active site of small enzymes

(such as, HIV and SIV proteases) requires an

appro-priate large cavity which is provided at the subunit

interface upon oligomerization In other words, it

appears that the quaternary structure has a ‘structural

functionality’ for most of the small dimeric proteins

and enzymes ( 100 amino acids in length)

Dimerization controls the stability and ‘quality’

of class B proteins

At variance with the above discussed group, the folding of class B proteins is somehow more closely related to that of medium-sized monomeric proteins

In this case each monomer undergoes an independ-ent assembly process, leading to a rather stable monomeric intermediate that only dimerizes after (partial) folding Indeed it has been found that most (if not all) of these intermediates resemble the ‘mol-ten globule’ state found in the folding pathway of many single chain proteins

Due to their larger size, the percentage of amino acid residues present at the subunit interface ( 15%)

is on average much smaller than that observed for small size dimers ( 42%) Despite this lower contri-bution to the total buried surface (Fig 4B), the dimeri-zation plays a fundamental role in the stabilidimeri-zation of class B proteins This is illustrated in Fig 6, where the free energy of dissociation (DG1) is compared to that

of the monomers unfolding process (DG2) It is shown that DG1 accounts for more than 60% of the total sta-bilization energy for half of the proteins considered and 50% for most of the others This contribution might arise from a tighter interaction between the sub-units in the dimer of class B Indeed, an analysis of the crystallographic data shows that in this group of pro-teins, water is hardly present at the dimeric interface More than 50% of the dimers in class A have been found to contain solvent molecules entrapped within the two dimers (data not shown)

In conclusion it appears that the role of dimerization for proteins of class B is mainly structural However, it

is quite clear that an early dimerization of partially

-0.80

-0.40

0.00

0.40

aa/subunit

Fig 5 Hydropathy at the subunit interface for class A dimers (h),

B (d) and C (m).

0.2 0.3 0.4 0.5 0.6 0.7 0.8

aa/subunit

Fig 6 Relative free energy of stabilization for a two-step unfolding process of class B dimers The percentage energy due to dimeriza-tion (DG 1 ⁄ DG TOT ) and to the monomers unfolding energies (DG 2 ⁄ DG TOT ) is reported as filled and unfilled symbols, respectively.

folded monomeric intermediates may reduce the risk

of formation of wrong aggregates

Assembling a dimer that lacks monomeric

intermediates: different folding roles

of quaternary structure?

According to a recent theory, protein folding can be

considered a biased search for the native state on a

rough potential energy surface that represents all the

possible tridimensional conformations [19,20]

Especi-ally in the case of larger proteins, this search may not

be unidirectional This means that the unfolded

poly-peptide chains, which populate the disordered unfolded

state, can reach the folded conformation through

dif-ferent pathways, which are characterized by difdif-ferent

local minima Depending on the energy barriers that

confine these minima, partially folded intermediate

states may be populated, either facilitating the whole

process (‘on-pathway intermediates’) or trapping the

folding molecule in aggregated, misfolded

conforma-tions (‘off-pathway intermediates’) The folding of a

dimeric protein is even more complex, requiring at

some point a bimolecular reaction (the monomers

association) which may take place before, during or

after the formation of secondary and tertiary structure

in each subunit Theoretical models [21,22] predict that

the folding rate of monomeric proteins decreases not

only with the protein size but also with the number of

long-range contacts, i.e interactions among residues

that are far away in the primary structure

It is conceivable that an early interaction between the

nascent monomers may lead to a kinetic bonus in the

folding pathway of dimers, thus, significantly reducing

the degree of freedom of each polypeptide chain In

other words, the biased search for the final

conforma-tion might be facilitated by a significant reducconforma-tion of the

potential energy surface roughness upon dimerization

The characterization of stable dimeric intermediate

states during folding could be very important to test

this hypothesis Unfortunately, the presence of

second-order kinetics and possible competitive aggregation

processes (that act as kinetic traps) make this

experi-ment particularly difficult However, a

semiquantita-tive, topological analysis of the dimeric proteins

considered so far might help to find a possible

correla-tion between their size and sequence and quaternary

structure For this reason we considered the following

two parameters: interface amino acids range (IAR),

which represents the distance (i.e number of residues)

between the first and last amino acids that take part in

the intersubunit contacts (Fig 7, upper panel); and

squared loop length (SLL), which is the sum of the

squared distances (in amino acid residues) between two successive residues of the primary structure involved in quaternary interactions (Fig 7, upper panel)

The two parameters have been normalized to the length of the monomeric subunit (n) and to n2, respectively, so that they both vary between 0 and 1 The meanings of IAR and SLL are better clarified in the examples reported in Fig 7 (1, 2 and 3), represent-ing three simplified models of the possible quaternary topologies in homodimers The values obtained for class A and class C dimers are reported in Fig 8 as a function of subunit length Both data sets are charac-terized by a decrease of the IAR parameter with pro-tein size, while SLL increases initially and, after reaching a maximum, falls back to lower values Inter-estingly, these values for class B dimers do not follow any regular pattern (data not shown) Comparing this behavior with the DG data reported in Fig 3A,C, indicates that the highest stability of medium-sized

Fig 7 (Upper panel) Cartoon illustrating the parameters IAR and SLL (Lower panel) Representation of three possible quaternary conformations assumed by dimeric proteins (the dimeric interface

is shown in red) The typical IAR and SLL corresponding values obtained are reported for each case The green circles and blue squares represent the N- and C-terminals respectively.

proteins of both classes A and C (i.e. 100 and  350

amino acids per subunit, respectively) is achieved with

large values of IAR and SLL (Fig 7, model 2)

Smal-ler and larger dimers show large or medium IAR but

small SLL (Fig 7, models 1 and 3, respectively) These

findings suggest that the quaternary structure gives a

different contribution to the folding process, depending

on the dimer size Folding is driven by a minimization

energy search that involves both protein and solvent

(water) molecules Small and medium sized dimers

(£ 100 amino acids per subunit) all show a high

con-tent of secondary structure (‡ 60%) and a high

inter-face hydropathy (Fig 5) It can be argued, therefore,

that the gain in the stabilization energy upon folding,

DG < 0, may arise from two quite distinct sources: (a)

a large increase of local interactions (DH  0), due to

the formation of a-helices and b-sheets; (b) a relevant

increase of the system entropy (DS  0), arising from

the hydrophobic effect at the subunit interface The

last effect probably replaces the early, entropy-driven

hydrophobic collapse that leads to the molten globule

states of monomeric proteins [23] In contrast, the

sta-bilization mechanism of larger dimers appears to be

quite different They have a significantly smaller

per-centage of secondary structure (on the average less than 40%), which probably reduces the enthalpy con-tribution to stability, and a less hydrophobic dimeric interface (Fig 5), suggesting also a smaller contribu-tion of quaternary interaccontribu-tions toDS

This is only partially counterbalanced by the pres-ence of hydrogen bonds, salt-bridges and other polar interactions at the subunit interface, explaining the decrease in the DG beyond certain molecular mass values reported in Fig 3

In conclusion we believe that the large number of homodimeric proteins found in living systems does not occur by chance For class B dimers, the dimeri-zation process might find a rationale in the protec-tion and stabilizaprotec-tion of those molten globule states that alone are not able to complete their self-assem-bly process When a quasi-native monomeric inter-mediate is not formed, a role of dimerization in the assembly process is less understandable, but we sus-pect that it is an important way of making the fold-ing of proteins correct and fast In other words, both early interacting unfolded monomers (class A and C) and partially folded monomers (class B) may act as chaperones for their partners However, the experimental proof for this hypothesis will require the careful study of denaturation–renaturation of dimeric proteins under experimental conditions (vis-cosity, molecular crowding, presence of chaperones) more similar to the in vivo folding milieu Very recently, dimeric folding intermediates have also been found in the folding pathway of small DNA binding proteins [24,25] where they are also thought to play

a critical functional role [25] This finding not only underlines the great importance of partially folded oligomeric structures but also demonstrates that their presence in the protein folding world might be much more common than found up to now

Experimental procedures

A list of the dimeric proteins considered in this study is shown in Table 1 according to the specific unfolded path-way reported in the literature

The dimeric interface and the gap volume at the dimer interface have been evaluated using the ‘Protein–Protein’ Interaction Server (http://www.biochem.ucl.ac.uk/bsm/PP/ server/) [26] Hydropathy at the dimeric interface was evaluated according to the amino acid hydropathy scale reported by Kyte and Doolittle [16] In particular, the hydropathy of each amino acid side chain was weighted by its specific interface accessible surface area (provided by the

‘Protein Protein’ Interaction Server) and their sum

0.20

0.40

0.60

0.80

1.00

0.00 0.05 0.10 0.15 0.20 0.25

aa/subunit

0.20

0.40

0.60

0.80

1.00

0.00 0.05 0.10 0.15 0.20 0.25

aa/subunit

class “C”

class “A”

Fig 8 IAR (d) and SLL (s) value of class A (upper panel) and class

C (lower panel) dimers The dashed, dotted and gray areas

corres-pond to the interface models (1, 2 and 3) shown in Fig 7.

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