Báo cáo khoa học: The importance of being dimeric pptx

REVIEW ARTICLE The importance of being dimeric Giampiero Mei 1,2 , Almerinda Di Venere 1,2 , Nicola Rosato 1,2 and 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 different 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 homodimers 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. 16 FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 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 proportional 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, tetramers, pentamers, hexamers, 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). G. Mei et al. The importance of being dimeric FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 17 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, N 2 « 2U; class B, N 2 « 2I « 2U; class C, N 2 « I 2 « 2U where N 2 represents the native state, I and I 2 are 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 )1 for 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 0 200 400 600 800 1000 aa / subunit ∆G u /aa (kcal/mol) 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. The importance of being dimeric G. Mei et al. 18 FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 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 database code Source Reference Class A Dimerization domain of 32 11.5 Rat liver [27] Transcription factor LFB1 Troponin C LFIL 34 11.0 Skeletal muscle [1] a 2 (PRR) 35 12.8 Artificial [1] Arc repressor 53 11.0 1arq Bacteriophage p22 [28] bZip transcription factor GCN 56 13.5 Rat liver [29] Dihydrofolate reductase truncated 62 11.3 R plasmid [30] Repressor of primer Rop 63 17.2 1rop Escherichia coli [1] Cro repressor 66 11.2 1cro Bacteriophage lambda [31] Archeal histones 69 14.0 Methanobacterium formicicum [32] Dihydrofolate reductase 78 13.9 R plasmid [30] Papilloma virus strain 16E2 80 9.8 1a7g Human [33] Gene V 87 16.3 1vqb Bacteriophage f1 [34] HIV-1 protease 99 14.0 1a8g Human [35] SIV-1 protease 99 13.0 1siv Human [35] bc-crystallin S3A 103 19.0 1hdf Physarum polycephalum [36] TRP aporepressor 107 18.8 1wrp E. coli [37] Mannose-binding lectin 110 13.1 Garlic bulb [38] Subtilisin inhibitor 113 6.0 3ssi Streptomyces [39] Nerve grow factor 118 19.3 Mouse [40] Core histones H2A-H2B 127 11.0 Chicken [41] b-Lactoglobulin 162 12.0 1beb Bovine [42] Growth hormone 191 27.8 Human [1] Glutathione S-transferase P 207 25.3 2gsr Porcine [1] Glutathione transferase A1-1 221 26.0 1ags Human [43] Glutathione S-transferase 226 26.0 1gta Schistosoma japonicum [44] Pea lectin 233 18.8 2ltn Pea seeds [45] Triosephosphate isomerase 248 19.4 1hti Human [46] Myo-inositol monophosphate 270 11.5 Pig [47] Tropomyosin 284 12.4 Chicken [48] Acid phosphatase 432 6.7 4kbp Red kidney bean [49] 4-amino-N-butyrate transferase 472 12.7 Pig liver [50] Alkaline phosphatase 580 17.3 Calf [51] Class B Four helix boundle protein (a 2 ) 2 62 14.3 Artificial [52] Light chain (LC8) of cytoplasmatic dynein 93 15.9 Drosophila [53] Lys49-phospholipase 121 17.2 1qll Bothrops jararacussu [54] Desulfoferrodoxin 125 34.6 1dfx Desulfovibrio desulfuricans [55] HSOD 153 20.2 1spd Human [56] PSOD 173 17.3 1yai Photobacterium leiognathi [56] cAMP receptor protein 209 18.6 1run E. coli [57] Thymidylate synthase 316 27.5 2tdm Lactobacillus casei [58] Tyrosyl-tRNA synthetase 319 27.8 1tya Bacillus stearothermophilus [59] Prostatic acid phosphatase 354 11.0 1cvi Human [60] Creatine kinase 380 — 2crk Rabbit [61] Aspartate amino transferase 396 15.9 1aam E. coli [62] Methionine adenosyl transferase III 396 15.7 Rat [63] Alkaline phosphatase 580 12.7 Cod [51] Class C Histone-like HU protein 90 8.3 1mul E. coli [64] FIS 98 13.9 1ety E. coli [24] Hydrolase 336 40.4 1psc Organophosphorus [65] Luciferase 340 24.2 1luc E. coli [66] G. Mei et al. The importance of being dimeric FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 19 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 (DG diss ) of several dimer- ic proteins considered in this paper was obtained from equilibrium unfolding measurements. The DG diss 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 Database code Source Reference Prion Ure2 354 49.0 Yeast [67] Ascorbate oxidase 552 17.0 1aoz Green zucchini [68] SecA 901 22.5 E. coli [69] 0 10 20 30 0 100 200 300 400 500 600 0 100 200 300 400 500 600 0 10 20 30 40 B A C 0 20 40 0 400 800 aa/ subunit class “A” ∆Gtot (kcal/mol)class “B” ∆Gtot (kcal/mol)class “C” ∆Gtot (kcal/mol) 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. The importance of being dimeric G. Mei et al. 20 FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 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 0 100 200 300 400 500 600 DIS / ASA (per monomer) A 0 10 20 30 40 0 100 200 300 400 500 600 DIS /(bur surface) (%) 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 monomer 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). G. Mei et al. The importance of being dimeric FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 21 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 stabilization of class B proteins. This is illustrated in Fig. 6, where the free energy of dissociation (DG 1 ) is compared to that of the monomers unfolding process (DG 2 ). It is shown that DG 1 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 0 100 200 300 400 500 600 hydropathy (a.u) aa/subunit Fig. 5. Hydropathy at the subunit interface for class A dimers (h), B(d)andC(m). 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 100 200 300 400 500 600 aa/subunit ∆G (%) 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. The importance of being dimeric G. Mei et al. 22 FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 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 different 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 reduction 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 n 2 , 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. G. Mei et al. The importance of being dimeric FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 23 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 interactions to DS. 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 stabilization 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 arbitrar- ily normalized within the range ()1 ⁄ +1). 0.20 0.40 0.60 0.80 1.00 0.00 0.05 0.10 0.15 0.20 0.25 0 100 200 300 400 500 SLL SLL aa/subunit 0.20 0.40 0.60 0.80 1.00 0.00 0.05 0.10 0.15 0.20 0.25 0 100 200 300 400 500 600 aa/subunit class “C” class “A” IAR IAR 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. The importance of being dimeric G. Mei et al. 24 FEBS Journal 272 (2005) 16–27 ª 2004 FEBS References 1 Neet KE & Timm DE (1994) Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation. Protein Sci 3, 2167–2174. 2 Hubbard JS, Gross KH & Argos P (1994) Intramolecu- lar cavities in globular proteins. Protein Eng 7, 613–626. 3 Williams MA, Goodfellow JM & Thornton JM (1994) Buried waters and internal cavities in monomeric pro- teins. Protein Sci 3, 1224–1235. 4 Monod J, Wyman J & Changeux JP (1965) On the nat- ure of allosteric transitions: a plausible model. J Mol Biol 12, 88–118. 5 Cook RA & Koshland DE (1969) Specificity in the assembly of multisubunit proteins. Proc Natl Acad Sci USA 64, 247–254. 6 Weber G (1986) Phenomenological description of the association of protein subunits subjected to conforma- tional drift. Effects of dilution and of hydrostatic pressure. Biochemistry 25, 3626–3331. 7 Weber G (1987) Fluorescence spectroscopy at high pressure: techniques and results. In Current Perspectives in High Pressure Biology , pp. 235–243. Academic Press Inc. London. 8 Cantor CR & Schimmel PR (1980) Biophysical Chemis- try, W.H. Freeman & Co. San Francisco, CA, USA. 9 Yu EW & Koshland DE Jr (2001) Propagating conformational changes over long (and short) distances in proteins. Proc Natl Acad Sci USA 98, 9517–9520. 10 Uversky VN (2002) Cracking the folding code. Why do some proteins adopt partially folded conformations adopt partially folded conformations, whereas other don’t? FEBS Lett 514, 181–183. 11 Careri G, Fasella P & Gratton E (1979) Enzyme dynamics: the statistical physics approach. Annu Rev Biophys Bioeng 8, 69–97. 12 Koshland DE (1973) Protein shape and biological con- trol. Sci Am 229, 52–64. 13 Torshin IY (1999) Activating oligomerization as inter- mediate level of signal transduction: analysis of protein- protein contacts and active sites in several glycolytic enzymes. Front Biosci 4, d557–570. 14 Torshin IY (2002) Functional maps of the junctions between interglobular contacts and active sites in glyco- lytic enzymes – a comparative analysis of the biochemical and structural data. Medical Sci Monit 8, BR123–135. 15 Miller S, Lesk AM, Janin J & Chothia C (1987) The accessible surface area and stability of oligomeric pro- teins. Nature 328, 834–836. 16 Kyte J & Doolittle RF (1982) A simple method for dis- playing the hydropathic character of a protein. J Mol Biol 157, 105–132. 17 Lins L, Thomas A & Brasseur R (2003) Analysis of accessible surface of residues in proteins. Proteins Sci 12, 1406–1417. 18 Zhang L & Hermans J (1996) Hydrophilicity of cavities in proteins. Proteins 24, 433–438. 19 Dinner AR, Sali A, Smith LJ, Dobson CM & Karplus M (2000) Understanding protein folding via free-energy sur- faces from theory and experiment. Trends Biochem Sci 25, 331–339. 20 Brooks CL III, Onuchic JN & Wales DJ (2001) Taking a walk on a landscape. Science 293, 612–613. 21 Plaxco KW, Simons KT & Baker D (1998) Contact order, transition state placement and the refolding rates of single domain proteins. J Mol Biol 277, 985–994. 22 Ivankov DN, Garbuzynskiy SO, Alm E, Plaxco KW, Baker D & Finkelstein AV (2003) Contact order revis- ited: influence of protein size on the folding rate. Protein Sci 12, 2057–2062. 23 Tsai CJ, Maizel JV Jr & Nussinov R (2002) The hydro- phobic effect: a new insight from cold denaturation and a two-state water structure. Crit Rev Biochem Mol Biol 37, 55–69. 24 Topping TB, Hoch DA & Gloss LM (2004) Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation. J Mol Biol 335, 1065–1081. 25 Ramstein J, Hervouet N, Coste F, Zelwer C, Oberto J & Castaing B (2003) Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure. J Mol Biol 331, 101–121. 26 Jones S & Thornton JM (1996) Principles of protein– protein interactions derived from structural studies. Proc Natl Acad Sci USA 93, 13–20. 27 De Francesco R, Pastore A, Vecchio G & Cortese R (1991) Circular dichroism study on the conformational stability of the dimerization domain of transcription fac- tor LFB1. Biochemistry 30, 143–147. 28 Bowie JU & Sauer RT (1989) Equilibrium dissociation and unfolding of the Arc repressor dimer. Biochemistry 28, 7139–7143. 29 Thompson KS, Vinson CR & Freire E (1993) Thermo- dynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4. Biochemistry 32, 5491–5496. 30 Reece LJ, Nichols R, Ogden RC & Howell EE (1991) Construction of a synthetic gene for an R-plasmid- encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. Biochemistry 12, 10895–10904. 31 Jana R, Hazbun TR, Mollah AK & Mossing MC (1997) A folded monomeric intermediate in the forma- tion of lambda Cro dimer-DNA complexes. J Mo1 Biol 273, 402–416. 32 Li WT, Grayling RA, Sandman K, Edmondson S, Shriver JW & Reeve JN (1998) Thermodynamic stability of archaeal histones. Biochemistry 37, 10563–10572. 33 Mok YK, de Prat Gay G, Butler PJ & Bycroft M (1996) Equilibrium dissociation and unfolding of the G. Mei et al. The importance of being dimeric FEBS Journal 272 (2005) 16–27 ª 2004 FEBS 25 [...].. .The importance of being dimeric 34 35 36 37 38 39 40 41 42 43 44 45 26 dimeric human papillomavirus strain-16, E2 DNA-binding domain Protein Sci 5, 310–319 Liang H & Terwilliger TC (1991) Reversible denaturation of the gene V protein of bacteriophage f1 Biochemistry 30, 2772–2782 Grant SK, Deckman IC, Culp JS, Minnich MD, Brooks IS, Hensley P, Debouck C & Meek TD (1992) Use of protein unfolding... TO (1993) Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits J Biol Chem 268, 10773– 10779 67 Zhu L, Zhang XJ, Wang LY, Zhou JM & Perrett S (2003) Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer... mutants of trp repressor: effects on oligomerization conformation, ligation and stability J Mol Biol 264, 32–45 Bachhawat K, Kapoor M, Dam TK & Surolia A (2001) The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization Biochemistry 40, 7291–7300 Tamura A, Kojima S, Miura K & Sturtevant JM (1995) A thermodynamic... intermediate: iron cofactors dissociate after polypeptide unfolding Biochemistry 40, 4940–4948 56 Stroppolo ME, Malvezzi-Campeggi F, Mei G, Rosato N & Desideri A (2000) Role of the tertiary and quaternary structures in the stability of dimeric copper, zinc superoxide dismutases Arch Biochem Biophys 377, 215– 218 57 Malecki J & Wasylewski Z (1997) Stability and kinetics of unfolding and refolding of cAMP receptor... a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure J Mol Biol 331, 101–121 FEBS Journal 272 (2005) 16–27 ª 2004 FEBS The importance of being dimeric 65 Grimsley JK, Scholtz JM, Pace CN & Wild JR (1997) Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate Biochemistry 36, 14366–14374... the conformational and dimeric stabilities of HIV-1 and SIV proteases Biochemistry 31, 9491–9501 Kretschmar M & Jaenicke R (1999) Stability of a homo -dimeric Ca(2+)-binding member of the beta gamma-crystallin superfamily: DSC measurements on spherulin 3a from Physarum polycephalum J Mol Biol 291, 1147–1153 Reedstrom RJ, Martin KS, Vangala S, Mahoney S, Wilker EW & Royer CA (1996) Characterization of. .. Erhardt J, Sluis-Cremer N & Dirr H (1997) Conformational stability of pGEXexpressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag Protein Sci 6, 399–406 Ahmad N, Srinivas VR, Reddy GB & Surolia A (1998) Thermodynamic characterization of the conformational stability of the homodimeric protein, pea lectin Biochemistry 37, 16765–16772 G Mei et al... characterization of a monomeric intermediate Biochemistry 29, 1907–1913 63 Gasset M, Alfonso C, Neira JL, Rivas G & Pajares MA (2002) Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites Biochem J 361, 307–315 64 Ramstein J, Hervouet N, Coste F, Zelwer C, Oberto J & Castaing B (2003) Evidence of a thermal unfolding dimeric intermediate... J Mol Biol 328, 235–254 68 Mei G, Di Venere A, Buganza M, Vecchini P, Rosato ` N & Finazzi-Agro A (1997) Role of quaternary structure in the stability of dimeric proteins: the case of ascorbate oxidase Biochemistry 36, 10917–10922 69 Doyle SM, Braswell EH & Teschke CM (2000) SecA folds via a dimeric intermediate Biochemistry 39, 11667–11676 27 ... 660–669 58 Gokhale RS, Agarwalla S, Santi DV & Balaram P (1996) Covalent reinforcement of a fragile region in the dymeric enzyme thymidylate synthase stabilizes the protein against chaotrope-induced unfolding Biochemstry 35, 7150–7158 59 Park YC & Bedouelle H (1998) Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate A quantitative analysis under equilibrium . 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. energy of unfolding per residue for monomeric (d) and dimeric (s) proteins as a function of the total number of amino acids. The importance of being dimeric