Archives of Medical Research 33 (2002) 245–249

0188-4409/02 $–see front matter. Copyright © 2002 IMSS. Published by Elsevier Science Inc.PII S0188-4409(02)00355-7

REVIEW ARTICLE

Specialized Biology From Tandem

�

-Turns

Jaime Lagúnez-Otero,

a

Andrea Díaz-Villaseñor

b

and Venkatesan Renugopalakrishnan

c

a

Departamento de Fisioquímica, Instituto de Química,

b

Departamento de Biología Molecular, Instituto de Investigaciones Biomédicas,Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico

c

Children’s Hospital, Harvard Medical School, Boston, MA USA

Received for publication April 18, 2001; accepted October 24, 2001 (01/051).

Diverse forms of pathologies can be derived from the lack of flexibility in tissues and the ab-sence of required concentrations of certain types of proteins (e.g., amelogenesis imperfecta).

�

-spirals using canonical proline-nucleated

�

-turns in diverse proteins allow for vital func-tions including structural (mucin and amelogenin), respiratory (elastin), muscular (titin), andthat of genetic expression (RNA polymerase II). These confer particular physical and chem-ical properties to proteins and therefore to the tissues in which they are found, while the per-vasive presence of tandem repeats in the genome sequence indicates their importance. Thispaper discusses the general biomedical relevance of this structure, focusing on several pro-teins found in humans. © 2002 IMSS. Published by Elsevier Science Inc.

Key Words:

�

-turn,

�

-spiral, Peptidic etiologies, Elasticity of tissue, Amelogenesis imperfecta, Arte-riosclerosis.

Introduction

Tandem repeats in the genome have been found to occur ina number of protein sequences. They confer important struc-tural features and specialized functional roles for this diversegroup of proteins. Among their more interesting characteris-tics are those originating from the

�

-turn generating prolinein each of the repeated segments.

Proline has amino nitrogen cyclized with the side chainterminal. This greatly restricts the conformational space avail-able to the peptide in which it occurs, and depending on theirplacement in the tandem repeating sequence results in a re-duced barrier to cis-trans isomers of the peptide bond in thevicinity of the residue and a loss of hydrogen bonding capa-bility of the imino nitrogen.

The

�

-spiral (Figure 1) consists of a helical array of one ofthe variants of a

�

-turn, sometimes stabilized by one or moreH-bonds (1). Hydrophobic interactions among the sidechains constitute the dominant stabilizing force in the

�

-spi-ral structure.

�

-turns, originally proposed by Venkatachalam(2,3) and further elaborated by Lewis et al. (4), have now

been found to be a ubiquitous secondary structural motifamong known protein structures. Adoption of

�

-spirals as astructural motif allows for unusual functions not determinedby the more common

�

-helices and

�

-sheets.In tropoelastin and amelogenin, the repetitive sequence

comprising the

�

-spiral is an important feature of the sec-ondary structures. Detailed physico-chemical studies haveshown that both proteins manifest a phenomenon known asinverse temperature transition, which allows for a decreasein entropy on temperature increase; however, other proper-ties such as elasticity and load-bearing in tissues subjectedto constant stress and strain should be discussed (Figure 1).

This paper presents the following seven examples of pro-teins that exploit the

�

-spiral: 1) tropoelastin (5–7); 2) toothenamel protein (amelogenin) (8); 3) RNA polymerase II fromnon-prokaryotic or viral organisms (9–12); 4) prolamin stor-age proteins of wheat and related cereals (13); 5) titin (14);6) mucins (15,16), and 7) flagelliform silk cDNA (17).

Tropoelastin

The tandem repeats in elastin (18) have been ascribed a rolein its elastomeric property of elastic fibers. The most impor-tant repeating sequence, poly(VPGVG), occurs in bovine andporcine tropoelastin (6,19). The molecular conformation ofpoly(VPGVG) has been shown to consist of a repeating type

Address reprint requests to: Jaime Lagúnez-Otero, Ph.D., Investi-gación, Instituto de Química, UNAM, Circuito Exterior, CU, 04510 Méx-ico, D.F., México. Tel.: (

�

52) (55) 5622-4424; FAX: (

�

52) (55

)

5616-2217, 2203; E-mail: lagunez@servidor.unam.mx

246

Lagúnez-Otero et al./ Archives of Medical Research 33 (2002) 245–249

II

�

-turn with the 10-atom hydrogen-bonded ring occurringbetween Val 1, C

�

O, and Val 4 NH groups based on ex-tensive structural studies by Urry and co-workers (18,20).Poly(VPGVG) in aqueous solution wraps around to form ahelical

�

-spiral structure on raising the temperature, a phenom-enon known as inverse temperature transition (18), whereinsome components of the system diminish entropic contenton increasing the temperature. This implies that the systemas a whole appears to diminish entropy. The

�

-spiral struc-ture is stabilized by hydrophobic interactions, especially Val1

�

-CH 3 and Pro 2

�

CH 2 moieties. These can be denaturedat temperatures

�

60

�

C with loss of elastic force and of elas-tic modulus.

Amelogenin

Amelogenins are a group of extracellular hydrophobic phos-phoproteins that comprise the bulk of the proteins from mam-malian dental enamel. Amelogenin, a

�

19 to 20 kDa pro-tein from calf tooth enamel, contains a primary sequenceunique in known mammalian proteins. It is characterized bya large proportion of Leu, His, Met, and Pro residues and asingle phosphorylated serine residue at position 16. Amelo-genin structure/function studies have been the focus of re-search since 1984 at our laboratories (21–26). On examiningthe primary structure of amelogenin, it is rather surprising tofind that it is implicated in the building up of Ca

��

on a be-wildering time scale. The entire process of mineral accumu-lation is complete in the very early phase of mammaliantooth development, and amelogenin is rapidly degraded en-zymatically after initiating mineralization of enamel. Primafacie, an extremely hydrophobic protein such as amelogenin,sparse in traditional Ca

��

chelators viz. Glu/Asp residues,will not be a candidate in ferreting Ca

��

ions to help buildtooth enamel. The primary structure of calf amelogenin wasderived by Edman degradation-gas phase sequencing (27) andlater from its cDNA sequence (8,28). The structural studiesof bovine amelogenin were performed from the native-pro-tein extracted from calf tooth enamel; later, however, weswitched to molecular biological methods to produce bovineamelogenin from cDNA.

The conclusions of FT-IR and laser Raman studies (23,24)were corroborated by detailed 2D and 3D NMR studies ofbovine amelogenin in solution phase (data not shown). NOESYspectra of amelogenin reveal characteristic cross-peaks aris-ing from the tandem

�

-turns. Amelogenin is a protein rich in

�

-sheet:

�

-turn segments. Extensive protein secondary struc-ture prediction schemes—algorithms and neural network (25)—have predicted short

�

-helical segments occurring betweenthe following residues: 22–29; 42–49, and 60–65, and anN-terminal segment involving 163–170.

�

-Helical segmentsare present in calf amelogenin but are greatly diminished incomparison to

�

-sheet:

�

-turn structures.The absence of polar amino acid residues and the occur-

rence of a single phosphorylated Ser residue at position 16

suggested that the

�

-spiral functional domain must deriveits Ca

��

sequestering capability from neutral carbonyl groupsof the amino acid. Similar proposals for calcification of tro-poelastin have been advanced by Urry et al. (18), in whichthe neutral C

�

0 groups are involved in Ca

��

binding bycharge neutralization hypothesis. Quantum mechanical stud-ies of Ca

��

binding to neutral peptide groups surprisingly haveshown that cation binding to neutral C

�

0 groups is ener-getically favored. A cluster of C

�

0 groups aligned on theinner surface of a staircase-like structure formed by repetitive

�

-turns occurring contiguously from Gln

112

through Leu

138

,Gln

112

-Pro-His-Gln-Pro-Leu-Gln-Pro-His

120

-Gln-Pro-Leu-Gln-Pro-Met-(Gln-Pro-Leu)

3

-Gln-Pro-Leu

138

is presented as anideal but novel candidate for Ca

��

binding. The uniquenessof this structure, labeled as

�

-spiral, and its putative role asCa

��

binding domain occurring between Gln

112

throughLeu

138

were the bases for selecting it for detailed molecularmechanics and dynamics studies (1). Molecular mechanicsand dynamics studies revealed a class of dynamic structurefor

�

-spiral region that could be broadly categorized into

�

-spiral structure with a

�

1 Å diameter pore and one inwhich the pore was blocked by Gln side chains. The

�

1 Ådiameter pore fits into it nearly as an unhydrated Ca

��

ion.The previously mentioned class of structures is stabilized

by 4

�

-turn H-bonds and Gln side chain to Gln side chainH-bonds. The hydrophobic side chains radially project out-ward with the C

�

0 groups lining the interior symmetrically.Entry of Ca

��

ions causes a perturbation of the hydrogenbond network and results in a movement of Gln side chains,allowing the passage of Ca

��

ions across the open pore. Thefree energy (

F) of cation binding to neutral C

�

0 groupsshould be expected to compensate for the free energy (

F)of disrupting the H-bond network. While the phosphory-lated Ser (16) may play a facilitating role in the passage ofCa

��

ions, the major functional role is believed to reside inthe

�

-spiral segment (29). Obviously, a structural entity suchas

�

-spiral must be encapsulated by the remainder of theprotein framework to stabilize the radially projecting hydro-phobic side chains. Amelogenin is believed to be associatedwith lipids, which may constrain its conformation and prob-ably induce dehydration of hydrated Ca

��

ions. We havecarried out lipid incorporation studies of amelogenin usingphosphatidylcholine and phosphatidylethanolamine; amelo-genin incorporates into both lipids quite well, as revealed byleakage studies.

Therefore, it is conceivable that the tandem repeat acts asthe nucleus or template around which the remainder of thechain wraps to form the globular protein predicted from hydro-dynamic studies. We obtained the 27-residue polypeptide bysolid-phase synthesis, extending the 27-residue polypeptideto larger fragments by elongating the N-terminal and C-termi-nal and then investigating the conformation of synthetic pep-tides. Fortunately, solid-phase synthesis of small proteins isnow feasible, which will make available pure material in largequantities, unlike the microgram quantities isolated from calf

Tandem Repeats with

�

-spirals in Protein

247

tooth enamel. We have inferred from the three different decaytimes

, observed from fluorescence spectroscopic studies,that either amelogenin manifests three conformers equili-brating rapidly in aqueous solution, or the three Trp residuesmanifest on the surface of amelogenin contribute to the threedifferent decay times

, which further complements infor-mation obtained from spectroscopic studies and 2D NMRstudies. Initially, the 27-residue polypeptide containing the

�

-spiral segment was synthesized using solid-phase poly-peptide synthesis (data not shown). We isolated and purifiedthe 27-residue polypeptide after considerable difficulty. De-tailed NMR studies of the polypeptide in aqueous and non-aqueous solution are presently in progress. Initial studies aresuggestive of Ca

��

channel-like activity, especially in non-polar solvents such as dimethyl sulfoxide-D6. Additionally,we are in the process of synthesizing analogs in which oneor more Gln residues were substituted to prevent formationof the side-chain hydrogen bonds followed by investigatingstructure-activity relationships. Chemical modification of Glnresidues is expected to result in retardation of channel-likeactivity manifested by the 27-residue polypeptide. Synthetic27 residue polypeptide doped in a lipid is expected to beuseful in inducing formation of enamel in certain types of den-tal diseases, e.g., amelogenesis imperfecta, in which enamelformation is retarded. In addition, the synthetic polypeptidewill serve as a model for investigating channel dynamicsand novel experiments for use as a Ca

��

delivery system.Further, we have planned protein engineering studies to de-sign channels on similar concepts for other cations with po-tential biomedical and technologic applications.

RNA Polymerase II

Pro

�

-turns are an integral part of the RNA polymerase IIstructure. Its largest subunit contains an unusual carboxy-terminal domain (CTD) consisting of tandem repeats of theconsensus sequence YSPTSPS (9,10). The sequence is re-peated from 17 to 52 times in RNA polymerase II of differ-ent organisms with the exception of eukaryotic RNA poly-merases I or III, and prokaryotic or viral polymerases (11).It has been suggested that CTD may play a role in mRNAtranscription. CD and NMR studies of a 56-residue syntheticpolypeptide, (YSPTSPS) clearly revealed three interlocked

�

-turns located at Ser2-Pro3, Thr4-Ser5, and Pro6-Ser7 ineach repeat, giving rise to a system of 24 H-bonds stabiliz-ing the CTD domain of RNA polymerase II.

Peptides representing single repeat units of the carboxy-terminal domain (CTD) of RNA polymerase II

32

(Tyr-Ser-Pro-Thr-Ser-Pro-Ser-Tyr-NH2, 1) contain overlapping Ser-Pro-Xaa-Xaa beta turn-forming sites that permit their overallstructure to closely resemble members of the quinoxaline classof antitumor DNA bis-intercalators. CD and NMR spectro-scopic investigations confirmed the presence of type II beta-turns within each of i

�

2 Gly- or D-substituted peptides inaqueous solution. In addition, an examination of singly substi-

tuted peptides suggests that an increase in the population of

�

-turn structure within the amino-terminal Ser-Pro-Xaa-Xaasite also increased the formation of beta-turn structure in theunmodified carboxy-terminal. In comparison, Ser-Pro-Xaa-Xaa site substitution in the carboxy-terminal site did not in-fluence structure in the remaining portion of the peptide.

Prolamins—Cereal Storage Proteins

It is to be expected that storage proteins also have taken ad-vantage of tandem-embedded proteins, conferring the nec-essary properties to the storage requirements. Both S-poorand -rich prolamins contain numerous repetitive sequences(13,30). The S-poor prolamins comprise C-hordeins of bar-ley,

�

-gliadins of wheat, and

�

-secalins of rye characterizedwith unusual repetitive sequences. The central domains ofC-hordeins and

�

-secalins consist of repeats based on theconsensus octapeptide repeat motif Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln with longer range repeats of 32 residues spreadover four octapeptide segments present in

�

-secalins. In thecase of C-hordein, the structure is a stiff worm-like chaincontaining type II

�

-reverse turns. The consensus composi-tion of the repeat motif in these proteins is (4 Gln: 3 Pro:1 Phe).The studies have been focused on C-hordeins (29), becausethey are relatively easily obtained in a pure form from a mu-tant barley line, sparse in most other hordeins. S-rich prola-mins are characterized by short repetitive sequences of thefollowing: the tetrapeptide Pro-Gln-Gln-Xaa; Pro-Gln-Gln-Pro-Phe-Pro-Gln (

�

-gliadins and

�

-secalins); Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln (

�

-hordeins); Pro-Gln-Gln-Pro-Tyr (

�

-gliadins); Pro-Gln-Gln-Gln-Pro-Phe-Pro-Ser (low-molecu-lar-weight glutenin subunit of wheat), and Pro-Gln-Gln-Pro-(Xaa)(Xaa)(Xaa) (

�

-hordein).

Titin

Titin is a

�

3,000-kDa large filamentous protein of verte-brate-striated muscle; single titin molecules extend from theZ disc to the M line. In its I-band section, titin behaves ex-tensibly and is responsible for myofibrillar-passive tensionduring stretch. Details of the molecular basis of titin’s elas-ticity are still under investigation. The I-band titin Ig repeatsexpressed in the stiff cardiac muscle and those that are tis-sue-specifically expressed in more elastic skeletal musclesrepresent distinct subgroups. Sequences of the titin gene thatcode for the C-terminal region of the PEVK domain are con-served in genomes of a larger variety of vertebrates, whereasN-terminal PEVK sequences are more divergent (14).

Mucins

Several MUC2 peptides have been synthesized, and theirsecondary structure has been analyzed by circular dichroismand Fourier transform infrared spectroscopic studies, as well

248

Lagúnez-Otero et al./ Archives of Medical Research 33 (2002) 245–249

as by other methods (16). For the binding studies, a MUC2mucin-protein core-specific monoclonal antibody was usedin competition RIA experiments. The minimal size peptidefunctioning as epitope was peptide 18PTGTQ22. Within theimmunodominant 13TPTPTPTGTQTPTT26 region, Uray etal. (16) found that all peptides recognized by the 996 mono-clonal antibody adopted beta-turn secondary structure. Pep-tides 15TPTPTGTQ22 and 16PTPTGTQ22, containing themost prominent beta-turn(s), had the strongest immunore-activity. It was also observed that peptides with Pro on theirN-termini (16PTPTGTQ22, 18PTGTQ22) adopt a differenttype of beta-turn in TFE than peptides with Thr at their N-ter-minal. Based on antibody binding, molecular dynamics cal-culations, and secondary structure analysis, a model has beenproposed for the epitope structure of the MUC2 mucin tan-dem repeat (16).

Flagelliform Silk

The tensile strength and flexibility of the structure is best ob-served in silk. Orb web-weaving spiders rely on their aerialnets to entrap flying prey. A key mechanical feature of orb-web design is the high elasticity of the capture spiral. Thecloning of cDNA for flagelliform-gland silk protein (17),which forms the core fiber of the catching spiral, has shownthat like all silks the flagelliform protein is composed largely

of iterated sequences. The dominant repeat of this protein isGly-Pro-Gly-Gly-X, which can appear up to 63 times in tan-dem arrays. This motif likely forms Pro2-Gly3 type II

�

-turns,and the resulting series of concatenated

�

-turns are thoughtto form a

�

-spiral. We propose that this spring-like helix isthe basis for the elasticity of silk. The variable fifth positionof the motif (X) is occupied by a small subset of residues(Ala, Ser, Tyr, Val). Moreover, these X amino acids occur inspecific patterns throughout the repeats. This ordered varia-tion strongly suggests that with hydration,

�

-spirals formhydrogen-bonded networks that increase the elasticity offlagelliform silk. The self-assembly of flagelliform proteinmonomers into silk fibers may be promoted by

�

-spiral/

�

-spiral interactions. Additionally, the other two motifs in theflagelliform protein, Gly-Gly-X and a spacer that disruptsthe glycine-rich regions, may contribute to the alignment ofmonomers into fibers. The flagelliform protein cDNA hasbeen compared to the other members of the spider silk genefamily. Apparently, all spider silk proteins can be character-ized as sets of shared structural modules. The occurrence ofthese modules among the proteins is inconsistent with thephylogenetic relationships inferred from C-terminal regions.

Conclusions

The participation of proline in the structure-function rela-tionship in

�

-spirals is based on the unique physico-chemicalconstraints, hydrophobicity, and imino bonding of the ami-noacid. Often overlooked by the medical community, pep-tidic motifs are very important in conferring special propertiesin tissues, including thermodynamic, mechanical, structural(31), and ion-conducting properties. These allow for vitalfunctions in higher organisms, such as breathing, circulation,and reproduction. In the case of tropoelastin, the

�

-spiralconfers the elasticity required for functioning of the lungs,thereby maintaining proper functioning at relatively high tem-peratures.

Elastin is the insoluble, elastic protein of high tensilestrength found in intercellular spaces of the connective tis-sues of large arteries, trachea, bronchi, and ligaments. It isprobable that many of the geriatric pathologies are due to theimproper distribution of these types of proteins. Indeed, itwas proposed three decades ago (32) that calcification ofaortic elastin may be the beginning of chronic arteriosclero-sis. As for amelogenins, the

�-spiral confers the chelatingproperties required in building tooth enamel, which has hy-droxyapatite crystals embedded in the architecture; withoutthese crystals, amelogenesis imperfecta is manifested.

The set of properties conveyed by the seriated �-turn canonly be derived from a special set of aminoacids found in aparticular order within the turn (29). Explanations can nowbe found for the low-information content of multiple tandemrepeats detected by sequence analysis of the human genome.It is clear that many of these segments correspond to vitallyimportant peptidic structures.

Figure 1. Model of �-spiral in glutenin (Osguthorpe DJ, Parchment O,unpublished).

Tandem Repeats with �-spirals in Protein 249

AcknowledgmentsWe thank Ma. Eugenia de la Torre-Hernández for assistance in thepreparation of this manuscript. This work was supported in part byDGAPA-UNAM.

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