Folding of Right- and Left-Handed Three-Helix Proteins

DOI: 10.1002/ijch.201300146

Folding of Right- and Left-Handed Three-Helix ProteinsOxana V. Galzitskaya,*[a] Leonid B. Pereyaslavets,[a] and Anna V. Glyakina[a, b]

1 Introduction

The problem of protein self-organization is the focus ofcurrent molecular biology studies. Apart from its funda-mental significance, knowledge of the mechanism of pro-tein folding could be put to practical use, such as the de-velopment of drugs and the design of de novo artificialproteins with defined properties.[1,2] Folding disturbancein vivo, frequently accompanied by protein aggregation,in many cases underpins human diseases.[3,4] Small pro-teins usually fold quickly and without folding intermedi-ates (i.e., it is a single-stage process), and “single-stage”kinetics is observed, whereas larger proteins fold ata lower rate and metastable intermediates are often de-tected, that is, folding is a multistage process and multi-stage kinetics is observed.[5] The folding times of globularproteins differ by many orders of magnitude, from micro-seconds to seconds and even hours.[6]

Folding of proteins has been considered in numerousexperimental and theoretical studies.[7–36] A variety of the-oretical models have been employed in these simulations,including Go and all-atom models, either with implicit orexplicit solvents. Multiple sampling methods have beenused as well, including umbrella sampling[37] and replicaexchange molecular dynamics simulations.[38] For themodeling of protein folding using the method of molecu-lar dynamics simulations, a special-purpose machine,called ANTON,[39] was designed and constructed. Thismachine greatly accelerates the execution of such simula-tions, producing continuous trajectories of 1 ms long. Thishas allowed new insights into two fundamental processesin protein dynamics: protein folding and interconversionbetween distinct structural states of a folded protein.[22]

To understand the fundamental principle of proteinfolding and its mechanism, many experimental and theo-retical works have been devoted to the folding of smalla-helical proteins.[7–36] The aim of such studies was to

obtain the three-dimensional structure of the protein, theprotein�s folding rate, the folding pathway, and the fold-ing mechanism. An important question that is touchedupon is whether the modeling of protein folding can tellthe difference between the folding of proteins with simi-lar structures, but different folding mechanisms.

It has been shown that, among proteins of the samesize, a proteins have both the least number of contactsper residue and the fastest folding rates in comparisonwith proteins from other structural classes. At the sametime, a/b proteins have both the greatest number of con-tacts and the slowest folding rates.[32] Enhanced packingin proteins results in frustration in its folding land-scape.[40–42] Moreover, it has been shown that more com-pact (spherical) proteins exhibit slower folding.[32] Pro-teins with multistate kinetics, on average, are more com-pact (spherical) than proteins with two-state kinetics.[34,43]

The obtained result suggests that the compactness of theprotein structure (shape properties) is an important deter-minant of the folding kinetics. Rapid- and slow-foldingproteins have additional structural diversities that con-tribute to their differing folding rates. The barrier heightfor the folding of large proteins is defined by the size of

Abstract : We are the first to investigate the relationship be-tween protein handedness and the rate of protein folding.Our findings demonstrate that small three-helix, left-handedproteins are less densely packed and should result in fasterfolding than that of right-handed, three-helix proteins. At thesame time, right-handed, three-helix proteins have higher

mechanical stability than the left-handed proteins. Moreover,from our analysis we have revealed that bacterial three-helixproteins have some advantages in packing over eukaryoticright-handed, three-helix proteins, which should result infaster folding.

Keywords: evolution · chirality · kinetics · protein folding · protein models

[a] O. V. Galzitskaya, L. B. Pereyaslavets, A. V. GlyakinaInstitute of Protein ResearchRussian Academy of Sciences4 Institutskaya Str., PushchinoMoscow Region, 142290 (Russia)Phone: +74956327871Fax: +74956327871e-mail: [email protected]

[b] A. V. GlyakinaInstitute of Mathematical Problems of BiologyRussian Academy of Sciences, 4 Institutskaya Str.Pushchino, Moscow Region, 142290 (Russia)

Isr. J. Chem. 2014, 54, 1126 – 1136 � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1126

Review

the boundary between folded and unfolded phases in thetransition state (the radius of the cross section[34]). Thisboundary is larger for a spherical protein globule thanthat for an elongated one.[32,34] Therefore, the proteinshape expressed by different parameters could be an im-portant determinant of the protein folding kinetics andprotein folding type.

2 Experimental and Theoretical Folding of EightThree-Helix Proteins

The B domain of protein A (BdpA) and the villin head-piece are the most studied three-helix domains in experi-mental and theoretical studies. It has been shown experi-mentally that these proteins fold rapidly and in a two-state manner.[8,10] Many papers are devoted to the foldingof the villin headpiece.[10,13,14,16,17,21,23] The lowest Ca root-mean-square deviation (RMSD) between experimentaland simulated three-dimensional structures was 0.39 �for residues 2–34 (excluding residues 1 and 35).[16] All-atom molecular dynamics simulations with the explicitmodel of water showed that folding of this protein couldbe described as a two-stage process following a well-de-fined pathway. Initially, the C-terminal and middle a-heli-ces are formed and then the N-terminal a-helix.[16] Thefolding nucleus is located around residues Phe17 andPro21. The same pathway was observed in our study[27]

using Monte Carlo simulations.The Ca RMSD for the best-folded structure of the B

domain of protein A was 0.80 �.[19] The C-terminal a-helix formed first, and Phe31 might play a critical role inachieving the correct packing of the three helices.[19] Shawwith co-workers modeled the folding of the villin head-piece and the GA module of the albumin-binding domain(PAB) using the all-atom model for proteins and the ex-plicit model for water.[23] They obtained three-dimension-al structures for these proteins with Ca RMSD of 1.3 �for the villin headpiece and 3.3 � for the PAB. The calcu-lated folding time for the villin subdomain was 2.8 ms,[23]

in comparison with the measured time of 4.3 ms using thelaser temperature jump for the wild-type subdomain at300 K.[44] For the PAB protein, the theoretical time is3.9 ms,[23] in comparison with the experimentally measuredtime of 2.5 ms.[45]

In some cases, a simple model can be used to describeprotein folding. The calculated folding thermodynamicsof a simple off-lattice three-helix bundle protein model(the B domain of protein A) under equilibrium condi-tions shows the following experimentally observed pro-tein transitions: a collapse transition, a disordered-to-or-dered globule transition, a globule-to-native-state transi-tion, and a transition from the active native state toa frozen inactive state.[12,25] In some cases, simple modelscorrectly estimated the folding rates, but not the foldingpathways.[26]

Two mechanisms of protein folding were distinguishedin the literature, nucleation�condensation and diffusion�collision (the framework model), which are different pre-sentations of a common mechanism of protein fold-ing.[10,46,47] According to the nucleation�condensationmechanism, secondary and tertiary protein structures areformed simultaneously, but under the diffusion�collisionmechanism of folding the elements of secondary struc-tures are formed in the beginning and then are arranged

Oxana V. Galzitskaya graduated fromthe Moscow Institute of Physics andTechnology (1990, Honorary Diploma)and obtained a Ph.D. in biophysicsfrom the Institute of Theoretical andExperimental Biophysics, Pushchino, in1996. Leading scientist, Head of theBioinformatics groups at the Instituteof Protein Research Russian Academyof Sciences (since 2008). She has auth-ored about 130 scientific papers andreceived an award for OutstandingRussian Young Scientists in 1997 anda Doctor of Science from the Russian Science Support Foundation.She is a member of the Editorial Boards of Current Protein and Pep-tide Science, Open Biochemistry, Open Bioinformatics, and Open Jour-nal of Biochemistry. Her research interests include investigating pro-tein folding and misfolding, the prediction of protein disorder andstructures, the prediction of amyloidogenic regions and foldingrates, the mechanical unfolding of proteins, and studying Alzheim-er’s disease.

Leonid B. Pereyaslavets graduated fromthe Moscow Institute of Physics andTechnology (2005). He received his Ph.D in biophysics from the Institute ofProtein Research, Pushchino, in 2010.He is now working as a Postdoc in theLevitt lab, Department of StructuralBiology, Stanford University, on an ac-curate description of nonbonded inter-actions in macromolecules. His currentresearch interests are focused on theinvestigation of protein folding, proteinuniverse space, and the developmentand assessment of molecular forcefields.

Anna V. Glyakina received a Diploma inphysics (2006) from the Tula State Uni-versity, Russia, and a Ph.D. in biophys-ics from the Moscow State Universityin 2013. Since 2006, she has beena member of the Laboratory of Molecu-lar Dynamics at the Institute of Mathe-matical Problems of Biology, RussianAcademy of Sciences. Her research in-terests include protein physics, molec-ular dynamics, molecular biology, andbioinformatics.

Isr. J. Chem. 2014, 54, 1126 – 1136 � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.ijc.wiley-vch.de 1127

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http://www.ijc.wiley-vch.de

in space to form a tertiary structure. In this case, the nu-cleation�condensation mechanism takes place when thesecondary structure is not stable in the absence of long-range interactions, at the same time, the diffusion�colli-sion mechanism becomes more probable with increasingsecondary structure stability.[11]

Gianni et al.[11] demonstrated that the right-handeddomain (the definition of domain handedness is given inFigure 1 and ref. [27]) of protein En-HD folded through

the framework model, whereas the right-handed domainof c-Myb folded through a mixed framework/nucleation�condensation model with a high-energy intermediate.Moreover, the right-handed domain of protein hTRF1[11]

folded through the nucleation�condensation mechanism.That is, for the right-handed domains, we see that, if a pro-tein folds under the diffusion�collision (framework)model, it folds faster than proteins which fold under thenucleation�condensation model.

For the right-handed domains of proteins, the proteinfolding rate is connected to the predisposition of aminoacid residues in the protein to form a secondary struc-ture.[11] For example, studies of three a-helical proteins(En-HD, TRF1, and c-Myb) revealed that the probabilityof the helical-state formation is larger for En-HD andsmaller for TRF1, protein c-Myb is situated between theabove-mentioned domains. This correlates with the fold-ing rates: En-HD folds faster than all other considereddomains and TRF1 folds slowly.[11]

Later, it was shown experimentally that the left-handeddomains of proteins PAB and BdpA were two of the fast-est folding proteins.[45,48] The PAB protein folds under thenucleation�condensation mechanism. Moreover, thedouble mutant of PAB (1prb7-53, K5I/K39V) exhibits thefastest folding rate known to date.[45] Second, BdpA foldsextremely rapidly in a two-state manner, with a time of8 ms.[8,48] The diffusion�collision theory (preorganized sec-

ondary structure) predicts folding and unfolding rate con-stants that are in good agreement with the experimentalvalues. This means that the mechanism of protein foldingcannot explain the difference between the folding ratesfor left- and right-handed proteins of a similar size. Also,it should be noted that there was no correlation betweenthe stability and folding rate in water both either theright-handed (homeodomain-like) domains[11] or the left-handed domains of proteins[8,45] (see Table 1; the correla-tion coefficient is �0.38).

The difference between folding rates in the three-helixdomains of proteins may be explained by the fact thatthey have different handedness. From experimental stud-ies, we conclude that left-handed, three-helix proteins(the GA module of the albumin-binding domain (PAB),the B domain of protein A (BdpA), the peripheral subu-nit-binding domain (psbd41), and the villin headpiecesubdomain (HP36)) fold faster than the right-handedones (the engrailed homeodomain (En-HD), the c-Myb-transforming protein (c-Myb), the RAP1 Myb domain(RAP1), and the TRF1 Myb domain (TRF1); see Table 1and Figure 2). Despite the large number of publicationsdevoted to three-helix folding, the role of handedness of

a-helices in protein folding is not yet understood. We arethe first to investigate the influence of this parameter onthe rate of protein folding.[27,28]

What might explain the difference in the folding ratesfor left- and right-handed proteins? We modeled the fold-ing of these three-helix proteins using Monte Carlo anddynamic programming methods.[27] The general resultfrom our simulations was that the left-handed domainsfolded faster than the right-handed domains (Table 2 andFigure 2).[27] Structural analysis of these proteins demon-strated that the left-handed domains had fewer contactsper residue and a smaller cross-sectional radius[34] thanthose of the right-handed domains (Table 2). This may beone explanation for the observed fact.[27]

Figure 1. Definition of handedness. Domain handedness was de-fined with the help of five points: three centers of helices (A, B,and C) and two midpoints between the corresponding edges ofhelices (ab and bc). Two torsion angles were calculated: shown inthe figure as forward- {A-ab-B-C} and back-propagated {C-bc-B-A}.If the signs of these torsion angles are equal, the quasi-torsionangle is defined as an average.

Figure 2. Experimental folding rates for four right-handed andfour left-handed a-helical proteins.


Review


3 Structural Properties of 332 Three-Helix ProteinDomains

It should be mentioned that the properties of the above-mentioned eight domains are transferable to all known

three-helix protein domains from the SCOP database(version 1.75).[49,50] Both the smaller number of contactsper residue and the smaller cross-sectional radius havealso been observed for the left-handed domains (less than60 amino acid residues), in comparison with those of the

Table 1. Different parameters for four right-handed and four left-handed a-helical proteins.

Name of protein(PDB entry/Number of residues) source

Three-dimensionalstructure

ln(kwater) in water/ln(kmt)in point of equilibrium(DGexp, kcal/mol)

Flexible re-gions

Radius of cross section (V/SASA)/Abs(CO) (Number of atom�atom contacts per residue withcontact distance rcont =6 �)

Left-handed

PAB (1prb/47)Peptostreptococcus magnus

13.8[7]/13.3(0.5�0.1)

2–912–1827–32,21 residues

3.14/580.0(74)

BdpA (1bdd/49)Staphylococcus aureus

11.7[8]/5.8(4.8�0.1)

15–2027–3540–49,25 residues

3.14/522.1(76)

psbd41 (2pdd/41)Bacillus stearothermophilus (Geobacillusstearothermophilus)

9.8[9]/9.8(2.64�0.06)

9–1421–36,22 residues

2.98/469.6(57)

HP36 (1vii/36)Gallus gallus (Chicken)

9.4[10]/10.6(2.46�0.06)

27–33,7 residues

2.99/403.0(76)

Right-handed

En-HD (1enh/54)Drosophila melanogaster(Fruit fly)

10.5[11]/8.1(1.70�0.02)

–0 residues

3.49/736.2(86)

c-Myb (1gv2/47)Mus musculus (Mouse)

8.7[11]/3.1(4.17�0.07)

5–932–38,12 residues

3.37/578.1(81)

RAP1 (1fex/59)Homo sapiens (Human)

8.2[11]/3.9(3.12�0.06)

19–3037–41,17 residues

3.42/769.6(71)

TRF1 (1ba5/49)Homo sapiens (Human)

5.9[11]/1.6(2.82�0.05)

10–1521–26,12 residues

3.45/711.6(83)


Review


right-handed domains from a data set of 332 a-helical do-mains of proteins (see Figure 3).[27] The cross-sectionalradii were 2.93�0.02 and 2.84�0.02 for the right- andleft-handed proteins, respectively.

Because the number of contacts depends on the size ofthe protein, the data set was divided into two subsets. Inthe first one, there were domains with lengths from 35 to60 residues (96 right-handed and 53 left-handed); in thesecond one, the domains lengths were from 61 to 150 resi-dues (142 right-handed and 41 left-handed; see Figure 3).

Such differences in the number of contacts for left- andright-handed proteins result from the enhanced tendencyof the side chains to fully interdigitate due to their posi-tioning in the right-handed a-helical scaffold, but notfrom the way pairs of helices interact. Packing angles be-tween two helices in known structures cover the fullrange from �180 to 1808,[51,52] but there are preferred ori-entations of two helices that are able to create the mostfavorable energetic interaction between side chains.There are four preferred angles: 1758 for the packing pat-

tern of an antiparallel right-handed coiled coil, �508 forthe right-handed parallel helix dimer, �1658 for the can-onical packing pattern of the left-handed antiparallel a-helix dimer, and 258 for the left-handed parallel coiled-coil pattern of the helix packing. The knobs-into-holesmodel is limited to helical coiled coils; it describes thecanonical heptad repeat coiled coils at �160 and 308.[53]

The ridges-into-grooves model describes the canonical a-helix packing at �50 and 1308.[54] However, these modelsonly describe interhelical packing. Globular a-helical pro-teins are “quasi-spherical” complexes with ball-like coresand their structures are not described by simple “layer”or “bundle” models. The problem of a simple representa-tion of the architecture of a-helices has been resolved bysuggesting the quasi-spherical polyhedron model.[55] Thepacking of n helices is described by a polyhedron with 2nvertices.

The calculated interhelical angles for our 332 three-helix protein domains cover the full range from 0 to 1808,as reported previously (see Figure 4).[51,52] The distributionof quasi-torsion angles (see Figure 5) with an average tor-sional angle of about 708 for the right-handed and �708for the left-handed domains demonstrates that we havea sufficient number of proteins: right-handed proteinshave representatives from 34 folds, among which there isthe only representative from 18 folds, and left-handedproteins have representatives from 22 folds, among whichthere is the only representative from 13 folds. For thequasi-spherical polyhedron model for three helical pro-teins, we have torsion angles of �728 for left-handed do-mains and +728 for right-handed packing of three a-heli-ces. One can see that this angle is close to the averageangle in our distribution (see Figure 5). This shows thatwe have a sufficient number of proteins to obtain sucha distribution and the polyhedron ideal model can de-scribe the packing of real proteins, as shown in 1988 for10 domains with packing of 3 a-helices.[55]

The distribution of the length of the helices indicatesthat the average length is 11 amino acid residues for boththe left- and right-handed protein domains (see Figure 6).

Table 2. Different average parameters for four right-handed andfour left-handed a-helical proteins.[a]

Parameters Left-handed Right-handed

< ln(kwater)> 11.5�0.9 8.3�0.9< ln(kmt)> 9.6�1.5 4.2�1.4< t1/2> ·104, Monte Carlo steps 9�3 61�13< tuf> , ps 291�106 541�43<Fmax> , pN v=0.01 �/ps 735�56 831�42

v=0.05 �/ps 831�37 928�39v=0.10 �/ps 879�53 1014�68

Number of contacts per residue (6 �) 70.9�4.6 80.2�3.2V/SASA 3.06�0.04 3.43�0.03

[a] ln(kwater) and < ln(kmt)> are logarithms of experimental foldingrates of proteins in water and in the midpoint of the transition; t1/2

is the folding time expressed in Monte Carlo steps (Monte Carlomodeling); tuf is the unfolding time of the protein under the actionof external force (molecular dynamics simulations); Fmax is the maxi-mal force necessary for the unfolding of a protein at a certain un-folding velocity (molecular dynamics simulations). For a more de-tailed explanation of these parameters, see refs. [27] and [28].

Figure 3. Distributions of contact density in the right- and left-handed proteins: all proteins (A); proteins of less than 60 amino acids (B),and proteins of more than 60 amino acids (C). Each point corresponds to a number of atom�atom contacts per residue within a 0.5 �thick layer with the layer center at the indicated distance.


Review


4 Mechanical Properties of Left- and Right-Handed Three-Helix Protein Domains

Studying the mechanical stability of a protein providesvaluable information on the energy landscape underlyingthe folding/unfolding processes. Some proteins in the cellexperience mechanical deformations: polypeptides unfoldunder a stretching force applied at specific amino acidsand subsequently refold.[56–58] From experimental studies,we have learned that a-helical proteins are less mechani-cally resistant than b-sheet proteins.[59] To find differencesin the behavior of left- and right-handed three-helix do-mains under mechanical stretching of these proteins, westudied the mechanical properties of these eight domains,under stretching at a constant speed and at a constantforce, on the atomic level using molecular dynamics simu-lations.[28] The analysis of 256 trajectories from moleculardynamics simulations with explicit water showed that theright-handed three-helix domains were more resistantmechanically than the left-handed domains (Table 2).Such results were observed at different extension veloci-

ties studied (192 trajectories obtained at the followingconditions: v=0.1, 0.05, and 0.01 �/ps; T=300 K) andunder a constant stretching force (64 trajectories, F=800 pN, T=300 K). We can explain this by the fact thatthe right-handed domains have a larger number of con-tacts per residue. In all mechanical unfolding trajectories,we observed that the terminal helices began to destroythe first ones in all eight proteins.

Mechanical stability is associated with the contactorder.[60] In our case, the correlations between the aver-age maximal force, average time of unfolding, and abso-lute contact order (Abs(CO)) are shown in Figure 7. Thecorrelation coefficients in the first case (force andAbs(CO)) are higher (the correlation coefficient is 0.72)than those in the second case (time of unfolding andAbs(CO)) (the correlation coefficient is 0.66).

Analysis of the amino acid distribution in the right-(total 16569 residues) and left-handed (total 6088 resi-dues) proteins demonstrated that the right-handed pro-teins prefer longer charged residues (Arg, Glu, Lys) incontrast to the preference of short Asp in the left-handedproteins (Figure 8). With regard to the loop residues inthe proteins, which play a key role in the formation of thedomain topology, the most noticeable relative differencebetween different types of loop regions is observed forglycine (about 20%). This may be connected with the re-quirement for greater flexibility of glycine in the left-handed proteins. For eight proteins with known experi-mental data, one can see that the left-handed proteinshave more flexible regions (about half for BdpA) than

Figure 4. Distribution of interhelical angles for 332 three-helix pro-tein domains.

Figure 5. Distribution of quasi-torsion angles for 94 left-handedproteins and 238 right-handed protein domains. Quasi-sphericalpolyhedrons represent the left- and right-handed three-helix do-mains. According to our definition, this angle is �728 for the left-handed pattern, and 728 for the right-handed one, which corre-sponds to average values for the quasi-torsional angle distribu-tions.


Review


the right-handed proteins (zero for En-HD; see Table 1),as calculated by using the FoldUnfold program.[61]

5 Folding of Bacterial and Eukaryotic Proteins

Questions concerning the evolution of protein structureshave been discussed in many works,[62] but nothing isknown about the evolution of the folding rates of proteinstructures. The need to maintain structural and functionalintegrity of an evolving protein severely restricts the setof allowable amino acid substitutions. The limits of pro-tein evolution were reported in a paper on the evolutionof protein sequences using data on the divergence of se-quences.[63] The authors showed that ancient proteins stilldiverged from each other, which was evidence of contin-ued expansion of the protein sequence “universe”. Inturn, the evolution of protein sequences has implicationsfor the evolution of the rates of protein folding.

Another difference between the right- and left-handedproteins is in the origin (Table 1). Four left-handed pro-teins belong to the kingdom of bacteria, at the same time,four right-handed proteins belong to the kingdom of eu-karyotes. Considering the known experimental foldingrates of 73 proteins (50 proteins following the “all-or-none” folding mechanism and 23 proteins folding with in-termediate accumulation, see the web site http://bioinfo.protres.ru/rate_evo_src_en.html), we concluded that bac-terial proteins with simple folding kinetics exhibiteda higher folding rate than that of eukaryotic proteins withsimple folding kinetics (see Table 3).[64]

Figure 6. Distribution of the lengths of a-helices (number ofamino acid residues) in the left- and right-handed protein do-mains.

Figure 7. Correlations between Abs(CO), average maximal force Fmax (A), and average time of unfolding tuf (B) for the left- and right-handeddomains.

Table 3. Mean value of ln(kwater) for bacterial and eukaryotic proteinsfollowing simple folding kinetics.

Bacteria Eukaryotes

All proteins 6.6�0.7(23 proteins)

4.9�0.7(27 proteins)

a 9.3�1.3(8 proteins)


b 5.8�0.5(8 proteins)


a+b 4.4�0.7(7 proteins)



Review


Can this observation be applied to all known three-helix protein domains[27] from the SCOP database? Weanalyzed structural domains within the kingdoms of livingorganisms in the SCOP database to see the protein distri-bution among the kingdoms. Upon database compilation,the main criterion for our selection was the quality of theprotein domain structure (structure with good resolution,the least number of ligands) rather than its origin. A totalof 3337 protein domains belonging to four main structuralclasses (a–d) with identities below 25% were found, in-cluding 727 a proteins of a class, 816 b proteins of b class,942 a/b proteins of class c, and 852 (a+b) proteins ofclass d. Figure 9 shows that eukaryotic proteins are better

represented in a-helical, b-structure, and a+b classes,whereas proteins of bacterial origin are more common inthe a/b and a+b classes. Archaean proteins are betterrepresented in a/b and a+b classes, whereas virus pro-teins are more abundant in the b structure class.

Table 4 demonstrates the distribution of 238 right- and94 left-handed three-helix proteins among different king-doms. One can see that most of the proteins belong tothe kingdoms bacteria (74 right- and 25 left-handed pro-teins) and eukaryotes (143 right- and 59 left-handed pro-teins).

Because bacterial proteins with simple folding kineticsexhibit a higher folding rate than that of eukaryotic pro-

Figure 8. Amino acid distribution in the right- and left-handed proteins and their loop regions.

Figure 9. Protein distribution in structural classes among kingdoms (A) and within the kingdom among structural classes (B).

Table 4. Distribution of right- and left-handed proteins within the kingdoms.

Kingdom All proteins Proteins length �60 amino acid residues Proteins length >60 amino acid residues

Left-handed(94)

Right-handed(238)

Left-handed(53)

Right-handed(96)

Left-handed(41)

Right-handed(142)

Archaea 6 10 1 5 5 5Bacteria 25 74 13 15 12 59Eukaryotes 59 143 37 70 22 73Viruses 4 10 2 5 2 5


Review


teins with simple folding kinetics, we propose that thenumber of contacts per residue for eukaryotic proteins ishigher than that for bacterial proteins. For 217 right- and84 left-handed a-helical proteins, we observed that theright-handed proteins from eukaryotes had a largernumber of contacts per residue than the right-handedproteins from bacteria (see Figure 10). For the left-handed proteins, such a tendency was not observed.

From Figure 11, one can see that the right-handed pro-teins from eukaryotes have a larger number of contactsper residue than the left-handed proteins from eukar-yotes. For bacterial proteins, the difference between theright- and left-handed proteins is only observed for largeproteins.

It is known that the bacterial protein synthesis occurs4–10 times faster than that of eukaryotic proteins.[65] Theaverage duration of the elongation cycle in bacterial sys-tems ranges from 0.05 to 0.1 s at 37 8C and is approxi-

mately three times greater at 25 8C. In eukaryotic systems,the rate of elongation is lower and varies widely due tomechanisms that regulate elongation. Typically, the aver-age eukaryotic elongation cycle is 0.1 to 0.5 s.[66] One cansuggest that, for proteins with simple folding kinetics, thein vitro folding rate correlates with the in vivo rate ofprotein synthesis. It is reasonable to suppose that smallprotein domains that do not require additional regulatorymechanisms will be synthesized at a higher rate in a bacte-rial system, whereas complex proteins will be more effi-ciently and rapidly synthesized in a eukaryotic system.

An interesting observation we have made is that theprobabilities for right- (238 three-helix domains) and left-handed domains (94 three-helix domains) to be the “firstdomain” in the existing protein are 0.447 and 0.574, re-spectively; therefore, this small anisotropy probably is as-sociated with cotranslational folding or other proteinproperties.

Figure 10. Distributions of contact density in the right-handed proteins from bacteria and eukaryotes: all proteins (A), proteins of less than60 amino acids (B), and proteins of more than 60 amino acids (C). Each point corresponds to a number of atom�atom contacts per residuewithin a 0.5 � thick layer, with the layer center at the indicated distance.

Figure 11. Distributions of contact density in the right- and left-handed proteins from eukaryotes: all proteins (A), proteins of less than 60amino acids (B), and proteins of more than 60 amino acids (C). Each point corresponds to a number of atom�atom contacts per residuewithin a 0.5 � thick layer, with the layer center at the indicated distance.


Review


6 Summary and Outlook

The understanding of chirality is an important field inmolecular biology both for protein structures and amyloidsuprastructures. The ability of a carbon atom to formchiral compounds is an important factor that determinesthe carbon basis of living systems on Earth.[67] Breakingof mirror symmetry in the molecular basis of life is appa-rently the first and one of the most striking examples ofsymmetry breaking in the natural sciences. Chiral asym-metry in the biosphere is uniquely realized at the geneticlevel and in biosynthesis, for example, l-amino acids andd-glucose.[68] For a-helical protein structures, we observesome preference in chirality on a different level of pro-tein organization. Chiral amino acids form chiral helicesthat self-assemble into structural domains: amino acidsare left-handed structures; the a-helix is a right-handedsecondary structure, which is the most regular and alsothe most prevalent. Finally, we found that the right-handed three-helix domains have a larger number of con-tacts per residues than the left-handed ones. This effect isthe result of the enhanced tendency of the side chains tofully interdigitate due to their positioning in the right-handed a-helical scaffold. During the past few years,many parameters have been proposed for the predictionof the rate of protein folding. We are the first to demon-strate that folding optimization depends not only on sec-ondary structure,[32,34] but on the handedness of a-helices.We observe some preferences in the packing of left-handed three-helix proteins both for bacterial and eu-karyotic proteins. Again, we can suggest that proteinshave evolved to pack more quickly and into looser struc-tures.[32]

Acknowledgements

This research has been performed with financial supportfrom the Russian Academy of Sciences (Molecular andCell Biology Program [01201353567] and FundamentalSciences to Medicine Program) and the Russian ScienceFoundation.

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Received: December 30, 2014Accepted: March 3, 2014

Published online: May 2, 2014


Review


Documents

Folding of Right- and Left-Handed Three-Helix Proteins