Inc.) on a MacIntosh IIfx (Apple Computer, Inc.) personal computer. Hydrophilicity was estimated using Kyte-Doolittle methods (20) with a window size of 7 to see ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Val. 267, No. 27, Issue of September 25, pp. 19320-19324,1992 Printed in W.S.A.
2 )
Isolation of a Clone Encoding a Second Dragline Silk Fibroin NEPHZLA CLAVZPES DRAGLINESILK
IS A TWO-PROTEINFIBER* (Received for publication, March 16, 1992)
Michael B. Hinman and RandolphV. Lewis$ From the Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071
Spider dragline silk is a unique protein fiber possessing both high tensile strength and high elasticity. A partial cDNA clone for one dragline silk protein (Spidroin 1) waspreviously isolated. However, the predicted amino acid sequence could not account for the amino acid composition of dragline silk. We have isolated a partial cDNA clone for another dragline silk protein (Spidroin 2), demonstrating that dragline silk is composed of multiple proteins. The amino acid sequence exhibits an entirely different repetitive motif than Spidroin 1. Spidroin 2 is predicted to consist of linked 8-turns in proline-rich regions which alternate with@-sheetregions composedof polyalanine segments. This structure for Spidroin 2 provides a model for dragline silk structure and function.
Spiders can generate several protein silks from specialized glands designed for diverse functions, unlike insects which produce only one type of silk. Nephila clauipes, an orb-web spinning spider, hassix different types of glands (1, 2), each producing a different silk. The presence of multiplesilks within a single organism presents a unique opportunity to examine protein structure and function. These silks are related by a predominance of alanine, serine, and glycine in their amino acid compositions. They are synthesized in specialized cells at the tail of their respective glands, secreted into a glandularlumen,and finally extrudedondemand through a duct and valve system ending ina spinneret. However, the different silks varywidely in mechanical properties, implying disparate aminoacid sequences. Severalfactors contributed to thechoice of dragline silk from the major ampullate gland of N . clauipes as the firstsilk to be studied, including: (i) its singular combinationof high tensile strength and high elasticity (3-6); (ii) ease of gathering large quantities of the silk (7); (iii) the size and accessibility of the gland; and (iv) previous extensive physical and chemical characterization (2, 8-11). Most structural models of dragline silk suggest a pseudocrystalline protein havingsignificant proportions of antiparallel P-sheet interspersed with regions of undefined structure which are thought to be responsible for the elastic properties (7, 12, 13). The amino acid sequence, which would give a great amount of information with regard to possible *This work was supported by Grant 28457-LS from the Army Research Office. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accessionnumber(s) M92913. $ To whom correspondence should be addressed Dept. of Molecular Biology, University Station, Box 3944, University of Wyoming, Laramie, WY 82071.
structure, was exceedingly difficult to obtain due to the solvent- and enzyme-resistant nature of dragline silk (10, 11). We made cDNA clones from the messenger RNA of the major ampullate glands in order to determine theprotein’s primary structure and gain insight into possible higher order structures. Thefirst cloneisolatedencodeda protein,Spidroin 1, containing repetitive elements which were not rigorously conserved in terms of sequence but didpreservea structural identity (14). Each structural repeat was composed of two segments, a polyalanine segment of 6-9 amino acids followed by a (Gly-Gly-Xaa), segment, where n ranged from 5 to 11. However, the sequence contained virtually noproline, known to be 3.5%of the aminoacid composition,and only two-thirds of the tyrosine found in N . clauipes dragline silk (11, 15). A pentapeptide, Gly-Tyr-Gly-Pro-Gly, purified from the acidcleaved silk at the same time as those used as the basis for cloning Spidroin 1 was not present in thepredicted sequence of Spidroin 1. MATERIALS ANDMETHODS
Isolation of the Spidroin 2 Clone-Twelve cDNA clones, in pBluescript (Stratagene), were isolated from a previously constructed library (14) using the oligonucleotide 5’-CCNGGNCCATANCC-3’ as a probe. The probe was labeled with 32P by kinasing (16) and hybridized to picked colonies (14) using the method of Wood et al. (17) employing tetramethyl ammonium chloride in the final 47‘C wash. Plasmid DNA was prepared,’ digested with EcoRI and BamHI, and subjected to agarose gel electrophoresis on a 1%agarose (Bethesda ResearchLaboratories) gel. The DNA was transferred to Zetaprobe (Bio-Rad) using a Vacublot technique (18).The resulting Southern blot was hybridized (17) with the same probe under the same conditions as the original colony screening. Four clones showed reasonable hybridization and pMASS2 was chosen because it contained the largest insert, approximately 2 kilobases. Nested Deletion Sequencing-pMASS2 was subjected to exonuclease I11 (Erase-a-Base kit, Promega) and used in a nested deletion strategy for sequencing. DNA from each time point was prepared using a quick plasmid preparation’ and subjected to double-stranded sequencing with Sequenase (United States Biochemicals) utilizing the 7-deaza-GTP sequencing kit (19). Each strand was sequenced at least two times, some sections as many as five times. This strategy helped resolve compressions seen during coding strand sequencing and aided in organizing the sequenced deletions, as the pMASS2 insert was highly repetitive at the nucleotide level as well as the peptide level. Computer Predictions of Hydrophilicity and Secondary StructureHydrophilicity profiles and secondary structure predictions were generated using MacVector, version 3.5 (International Biotechnologies, Inc.) on a MacIntosh IIfx (Apple Computer, Inc.) personal computer. Hydrophilicity was estimated using Kyte-Doolittle methods (20)with a window size of 7 to see smaller structure without background. Secondary structure was predicted using Chou-Fasman(21) algorithms and Robson-Garnier (22) algorithms separately, then generating a consensus prediction.
19320
’ Y. Kadokami and R. V. Lewis, manuscript in preparation.
Dragline Spider Silk
Two-protein IsFiber a
19321 90 30
CCTGGACGAZIATCCACCACCACAACAACCCCCAGGACCATATGCCCCTGGACAACAAGGACCATCTCCACCTCCCACTCCCCCTOQLGCA
P
G
G
Y
G
P
G
Q
Q
G
P
G
G
Y
G
P
G
Q
Q
G
P
S
G
P
G
S
A
A
A
A
C C A C C A C C C C C C C C A C C A C C A C C T C C A C C A T A T G C C C C T G G A C A A C A A G G A C C C C C A G G A T A T C C A C C A C C A C A A C A A C C A C C C C C A ~ 180 A A A A A A G P G G Y G P G Q Q G P G G Y G P G Q Q G P G R 60 270 90
TATCCACCACCACAACAACCACCATCTCGACCTCCCAGTGCCCCTGCACCCCCAGCAGGATCTCCACAACAACCCCCACGACGATATcca
Y
G
P
G
Q
Q
G
P
S
G
P
G
S
A
A
A
A
A
A
G
S
G
Q
Q
G
P
G
G
Y
G
CCACCPCAACAACCTCCA~CCTTATCCACAACCACAACAACCACCATCTCCACCAGcCACTCCACKX:~CCCTCACCCCCACCCTCA 360 P
R
Q
Q
G
P
G
G
Y
G
Q
G
Q
Q
G
P
S
G
P
G
S
A
A
A
A
S
A
A
A
S
l
2
CCACAAKTCCACAACAACCCCCACGACCTTATCCACCACCTCAACAAGGCCCACGAGCPTATGCACCA~TCAACAACCTCCTCCACCA A E S G Q Q G P G G Y G P G Q Q G P G G Y G P G Q Q G P G G 1
0
450 5 0
T A T C Q L C C A C C A C A A C A A G C a C C A T C T C G A C C A G G T A C T G C C C C P C C A G C A G C C C C C C C X : C C A T C A C C A C C T G G A C A A C A A ~ C C A C G A 540 Y G P G Q Q G P S G P G S A A A A A A A A S G P G Q Q G P G 1 8 0
C C A T A T ~ C C A C C T C A A C A A C C T C C T G G A G G A T A T G G A C C A G G A C A A C A A ~ C C A T C T C C A ~ ~ A C T C C C C C P G C A C C C C C C 630 CCC G Y G P G Q Q G P G G Y G P G Q Q G P S G P G S A A A A A A 210 L
720 240
TCTCCACCACCCACTCCACCTGCAGCAGCCGCAGCAGGACCTGGACAACAAGGACCCGGACCATATCCACCACCACAACAAGGACCATCT S G P G S A A A A A A A G P G Q Q G P G G Y G P G Q Q G P S
810 270
GCCCCATCACCACCTGGACAACAAGGACCAGGAGGATATGGACCAGGTCAACAAGGTCCAGGAGGTTATCCACCACCACAACAACQLCCA
A
A
S
G
P
G
Q
Q
G
P
G
G
Y
G
P
G
Q
Q
G
P
G
G
Y
G
P
G
Q
Q
G
C C A C C C C C T A G T G C C G c p G c A G c A G c a G c c G C C G C A ~ G G A C C T G G A G G A T A T G G c c c T ~ C A A C A A C C A C C C G c A ~ ~ T C Q L900 ccA G P G S A A A A A A A A A G P G G Y G P G Q Q G P G G Y G P 300
G G A C A A C A A G G A C C A K T c G A G C A G G c A G T G C A G C A G c A G c A G C C G C A G c A G G A C C T G G A C A A C A A ~ T T A G c A ~ T A T c G A C C A C C990 A
G
Q
Q
G
P
S
G
A
G
S
A
A
A
A
A
A
A
G
P
G
Q
Q
G
L
G
G
Y
G
P
G
330
C A A C A A C C T C C A C G A G G A T A T ~ C C A G G A C A A C A A C G T C C A G G A G G A T A T G G A C C A G C T A G T ~ ~ ~ C C A G C A C C C G C A C C A C1080 CA Q Q G P G G Y G P G Q Q G P G G Y G P G S A S A A A A A A G 360
CCTCCACAACAACCACCACCAGGATATGGACCTGGACAACAAGGACCATCTGGACCAGGCAGTGCATCTCCACCAGCACCCCCACCCCCA P G Q Q G P G G Y G P G Q Q G P S G P G S A S A A A A A A A
1170 390
GcAGGACCACCACCATATCCACCAGGACAACAAGGTCCAGGAGGATATGcaCCAGGACAACAAGGACCATCTGGACCAcccACTCCAZCT
1260 420
A
G
P
G
G
Y
G
P
G
Q
Q
G
P
G
G
Y
A
P
G
Q
Q
G
P
S
G
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G
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A
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G C A G C A C C A C C C C C A G C C ~ G C A G G A C C A G G A G G A T A T G G A C C A G G A C A A C A A G G T C C A G G A G G A T A T G C A C C A C C A C A A C A A C G A C C A 1350 A A A A A A A A G P G G Y G P G Q Q G P G G Y A P G Q Q G P 450 T C T C C A C C A G G C A C T C C A G C A G C A G C A ~ G C T G C T ~ G C A G G A C C T G G T G G A T A T ~ C C A C C C C A A C A C G G A C C A T C T C C T C C T C C1440 A
S
G
P
G
S
A
A
A
A
A
A
A
A
A
G
P
G
G
Y
G
P
A
Q
Q
G
P
S
G
P
G
480
A T C C C A C C P T C A G C T C C T T C A G C A G G A C C P G G A G G T T A T G G A C C A G C A C A A C A A G G A C C A G C T C G A T A T O C C C C T G G A A M : C C A G T A ~ 1530 I A A S A A S A G P G G Y G P A Q Q G P A G Y G P G S A V A 510
C C C K T C C C C C T G C A C C A T C P G C A G G T T A T G G G C C A C C T T C T C A A G C T T C C G C T G C A G C T T C T C C P C T G G C T T C T C C A C A T T C A C C C C C P 1620 A S A G A G S A G Y G P G S Q A S A A A S R L A S P D S G A 540 S A GTT GCA TCA GCT GTT KT AAC TTG GTA TCC AGT Gcx: CCA ACT AGC TCT
R
V
A
S
A
V
S
N
ATT GGC CCA AGT AAT CCT GGT CTC I G A S N P G L
L
V
S
S
TCT GGT TGC GAT S G C D
G
P
T
S
S
GTC CTC ATT CAA GCT V L I Q A
GCTGCC TTA TCA AGT GTT ATC AGT A A L S S V I S
S
S
I
G
Q
V
N
Y
G
A
A
S
Q
F
A
Q
N
CCT GTG KT CAA 1710 A V S Q 570
CPC TTG GAA ATC GTT TCT GCT TGT GTA ACC ATC CTT ZCT 1800 L L E I V S A C V T I L S 600
TCA TO2 AM: ATT GGT CAA GTT AAT TAT GGA GCG GCT TCT CAG TTC GCC CAA GTT
S
AAC
V
GTCGGC CAA TCT V G Q S
GTTTTG AGT GCA TTT TAA TTG M A 1890 V L S A F * 630
M T T T A T l l A A A A T A T C C A T G G A T T m : T A G C C T C G G C A A C T A A T T G C T C C T A C T A T G T A A T T T T T T T T T A B B T B B B T T ~ T c c A A C n c 1980
FIG. 1. DNA sequence of pMASS2 and predicted amino acid sequence of Spidroin 2. The termination codon, TAA, is marked with an asterisk. The putative polyadenylation signal site and the beginning of the poly(A)-tail are underlined. This sequence has been submitted to GenBank.
tail. The frequency of codon usage Spidroin in 2 (Table I) is to that of Spidroin i (14). For example, glycine, very similar The peptide absentfrom the Spidroin 1sequence, Gly-Tyrthe most prevalent amino acid in both proteins, shows an Gly-Pro-Gly, was used as a basis for designing a DNA probe 89% preference for Aor T as the thirdnucleotide in Spidroin (5’-CCNGGNCCATANCC-3’ ( n = A, T , C, or G))which was 2 and 94% in Spidroin 1. Glutamine shows a 97% preference used to rescreen the original major ampullate gland cDNA for A in Spidroin 2 and 98% in Spidroin 1. The large preferlibrary from which the Spidroin 1 clone had been isolated. A ence for A and T as the third nucleotide in dragline silk can second cDNA clone was isolated and sequenced which en- be explained by considering the secondary structure of the coded a separate, distinct protein, Spidroin2. mRNA. The repetitive nature of the DNA encoding the poTheDNA sequence isshownin Fig. 1 along withthe lyalanine segments,(GCX),, could result in numerous hairpin predicted amino acid sequence. The start of the 16-base long loops formed between nearby polyalanine coding regions if poly(A)-tail (nucleotide 1982) is shown underlined, as well as the third base were also G or C instead of A or T. The same the likely polyadenylation signal site (nucleotide 1961-1966). is true for other G/C-rich regions as well. Chavancy et al. (23) There is a relatively short 3“noncoding region with the stop have noted that in vitro translation of Bombyx mori mRNA codon (nucleotide 1882) occurring 97 bases before the poly(A)- proceeds more efficiently if the fibroin mRNA is heated to RESULTS
Dragline Spider Silk
19322
Two-protein IsFiber a TABLE I
Codon frequencies for Spidroin2 cDNA Codon (Amino acid)
TTT (F) TTC (F) TTA (L) CAT TTG (L) CAC TCT (S) TCC (S) CGT TCA (S) TCG (S) TAT (Y) TAC (Y) TAA (*) TAG (*) TGT (C)ATA TGC (C) ATG TGA (*) TGG (W) CTT (L) CTC (L) CTA (L) AAT CTG (L) CCT (P) ccc (P)
""_
Count
1 1 2 3 30 3 10 0 30 0 1 0 1 1 0 0
1 3 2 1 22 5
Codon (Amino acid)
Count
CCA (P) AGT CCG (P) (H) (H) CCA ( Q ) CAG (Q)GTC
59 0 0 0 72 2
(R) CGC (R) CGA (R) CGG (R) ATT (I) ATC (I) (I) (M) ACT (T) ACC (T) ACA (T) ACG (T) (N) AAC (N) AAA (K) AAG (K)
PGGYGPGQQGPGGYGPGQQGP--SGPGSAAAAAAAAAA GPGGYGPGQQGPGGYGPGQQGPGBYGPGQQGP--SGPGSAAAAAA---GSGQQGPGGYGPBQQGPGGYGQGQQGP--SGPGS"A
" " " " "
ESGQQGPGGYGPGQQGPGGYGPGQQGPGGYGPGQQGP--SGPGS--""""GPGQQGPGGYGPGQQGPGGYGPGQQGP--SGPGSAAAAAAAASGPGQQGPGGYGPGQQGPGGYGPGQQGL--SGPGSAAAAAAA--GPGQQGPGGYGPGQQGP--SGPGSAAAAAAAAA""""_ GPGGYGPGQQGPGGYGPGQQGP--SGAGSAAAAAAA--" " " " "
" " " " " " " " " "
GPGQQGLGGYGPGQQGPGGYGPGQQGPmGPGSASAAAAAA" GPGQQGPGGYGPGQQGP"SGPGSA3-
23
2 0 0 (A) 0 3 4 0 0 1 1 0 (G) 0 2 2 0 0
Codon (Amino acid)
(S) AGC (S) AGA (R) AGG (R) GTT (V) (V) GTA (V) GTG (V) GCT GCC (A) GCA (A) GCG (A) GAT (D) GAC (D) GAA (E) GAG (E) GGT GGC (G) GGA (G) GGG (G)
Count
17 3 2 0 7 2 3 1 32 75 2 2 0
2 0 20 19 143 2
ASAAASRLSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGAS Spidroin ASAAASRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGAS Spidroin
1
2
FIG. 3. Comparison of a conserved sequence found in the COOH-terminal regions of Spidroin 1 and Spidroin 2. Amino acids 646-694 of Spidroin 1 (from 14) are compared with amino acids 526-574 of Spidroin 2. Amino acids conserved in both sequences are in bold type.
" " " " "
" " " " " " " " " "
"_""" """"_
""""""""-""""
""""_
GPGGYGPGQQGPGGYDGQQGP--SGPGSASAAAAAAAA GPGGYGPGQQGPGGYuGQQGP--SGPGSAAAAAAZLAA-
GPGGYGPAQQGP--SGPGLAASAASA--GPGGYGPAQQGPAGY----------GPGSAYMAGA--
-------------------------csBGy"----------GpGS*AS"---
SRLASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVL IQALLEIVSACVTILSSSSIGQVNYGAASQFAQWGQSVLSAF
FIG. 2. Predicted amino acid sequence for the Spidroin 2 protein, rearranged to show repetitive elements. The most repetitive proteinelements have been arranged from theaminoterminal through the highly conserved region, followed by the less conserved region and divergent COOH-terminaltail.The dashes represent deletions, allowing the elements to be arranged for maximum identity.
10 alanines with an occasional conservative serine substitution. While the repetitive regions of Spidroin 1 and Spidroin 2 show very little homology, there is a 49-amino-acid region in the COOH-terminal domainof Spidroin 2 with 80% identity to a corresponding region in Spidroin1 (Fig. 3). These regions both follow the highly repetitive regions of their respective proteins. DISCUSSION
We believe Spidroin 1 and Spidroin 2 are the only two major protein subunits of dragline silk from N . cluuipes. All of the peptides reported ina previous paper (14), the peptide used for designing our probe (Gly-Tyr-Gly-Pro-Gly), andover 100 "C and quickly cooled, suggesting that a G/C-rich message may already havea great degree of secondary structure which 20 smaller peptides'produced by partial aciddigestion of would be compounded by having G or C in the thirdposition dragline silk are foundwithin the predicted protein sequences of Spidroin 1 and Spidroin 2. The total amino acid composiof the codons. The first 464 amino acids of the predicted partial protein tion of dragline silk can be accounted for by a combination of sequence can be grouped into highlyconserved repetitive the amino acid compositions of the two Spidroins. Using the elements, shown inFig. 2 with deviations from the conserved cloned cDNAsas probe^,^ each Spidroin exhibitsonly a single from the major ampullate sequence underlined. The next 65 residues show less conser- hybridizing mRNA band in extracts gland, indicating a unique mRNA for each protein. vation, and the last 98 residues are widely divergent. The The overall 3.5% proline content of dragline silk can be pentapeptide on which our probe was based, Gly-Tyr-Glyaccounted for by averaging the high level of proline (13.5%) Pro-Gly, is represented numerous times throughout the sein Spidroin 2 with thenegligible amount of proline in Spidroin quence. The proline-richregion has a repeating pentapeptide 1. Using their respective cDNAs as probes, the sizes of the motif in which Gly-Pro-Gly-Gly-Tyr alternates with Gly-Pro- mRNAs for Spidroin 1 and Spidroin 2 haverecentlybeen Gly-Gln-Gln. This region can bedivided into two distinct determined as 5.6 and 3 kilobases, respectively.s We calculated elements, a segment witha variable number of the alternating the aminoacid contribution of the two Spidroins to the total pentapeptides followed by a highly conserved core of 3 pen- amino acid composition of dragline silk by assuming that: 1) tapeptide repeats. The variations are groups of 5 amino acids, the length of each protein was proportional to thesize of its in contrast to the pattern seen in Spidroin 1 which consists of groups of 3 amino acids. The joining region, Ser-Gly-ProM. Xu and R. V. Lewis, unpublished results. Y. Kadokami and R. V. Lewis, manuscript in preparation. Gly-Ser, isvery highly conserved and leads into a series of 6-
Is a Two-protein Fiber
Dragline Spider Silk
19323
method confines any sheet-like structure to thehydrophobic mRNA; 2) the repetitive regions of both proteins extended into their respective uncharacterized amino-terminal regions; COOH-terminal tail. The consensusprediction (CfRg) shows in the proline-rich regions and that 3) themolecular ratio of Spidroin 1 to Spidroin 2 is that there are numerous turns about 3 to 2. The calculations indicate that Spidroin 1 and alternating withpolyalanine segments which are predicted to Spidroin 2 are sufficient to account for the total amino acid be helix-forming. We originally believed that thepolyalanine segments were composition. It should be noted that withina single strand of silk from one spider, the aminoacid composition varies from likely to form a-helices (lo), based on computer predictions site to site (11).Varying amounts of proline have also been for Spidroin 1and Spidroin2 and physical studies of peptides found in dragline silk from a single spider over its lifetime in aqueous solutions (10). Based on arguments which follow, (24).Thesevariations in amino acidcomposition can be we now believe that Spidroin 2 is composed of P-turn strucregions alternating with 0-sheet accounted for if the expression of the two Spidroin proteins turesintheproline-rich regions formed by the polyalanine segments. We also believe is independently regulated. The predominantuse of A or T (U) as the third nucleotide the polyalanineregions inSpidroin 1 are in the&sheet in the codonsfor glycine and alanine is notas pronounced as conformation. The @-turn structure, difficult to detect with x-raydiffracthat found for B. mori silk (25). However, it has been shown spectroscopy, or Raman specthat the production of dragline protein from spider silk mRNA tion, Fourier transform infrared is subject to translational pauses(26, 27) in the same manner troscopy, is the mostlikely conformation for the pentapeptide as B. mori silk (28) under bothi n vivo and i n vitro conditions. repeats of Spidroin 2 to adopt (32-34). Gly-Pro-Gly-Xaa-Yaa Both organisms appear to produce gland specific pools of repeats are known to form P-turns and a Gln in position 4 tRNAs forglycine and alanine as these amino acids represent may indicate type I1 P-turns (34). The P-turn conformation the major structural components of the silk proteins (23, 29, has been demonstrated for similar proline-rich sequences in 30). Efficient in vitro translation of the silk proteins from unrelated proteins such as gluten, synaptophysin, and elastin both organisms requires tRNA supplementation from appro- (32, 33). The polyalanine regions of Spidroins 1and 2 probably form priately conditioned glands (27, 28, 30). While the patternof tRNA accumulation inB. mori is approximately proportional the antiparallel 0-sheets observed by many studies (3-6, 8, to the use of corresponding amino acids (31), alanine tRNA 35). X-ray diffraction studies of drfgline silk from our labois the most abundant species in N . clavipes major ampullate ratory and others (36)show a 10.6-A spacing between stacks glands (30). Thedifference can probably be attributed to the of pleated sheets in dragline silk. This corresponds to the spacing found for 0-sheets formed by polyalanine (37). The different nature of the repetitive elements of the two silks. Since the alanines in both Spidroin 1 and Spidroin 2 are Gly-Gly-Xaa repeats of Spidroin 1 have been shown to be found in clusters of 6-10, as opposed to the dispersed nature unfavorablefor p-sheetformationin silk (38).Studieson of B. mori alanines ((Gly-Ala-Gly-Ala-Gly-Ser),), it may be polyalanine peptide crystals containinglow amounts of water necessarytohave alarger pool of alaninetRNAduring demonstrate only the presence of ,&sheet structures (39), and translation of spider silk to get through the areas of repetitive spider dragline silk contains less than 6%~ a t e rcorrespond,~ alanine usage. ing to less than one water molecule per 3 amino acids. The overall repetitive nature of the aminoacid sequence of Under tension, the linked @-turns in Spidroin2 may form Spidroin 2 isemphasized by computerpredictions of the a P-turn spiralor extend an already present spiral, as @-turns hydrophilicity and secondary structureof the protein(Fig. 4). are known to have a degree of structural flexibility (32, 33). This would be similar to an elastic mechanism proposed for Computerpredictions of secondarystructure usingChouFasman ( C F ) and Robson-Gamier (RG) statistical methods elastin (33) with entropic forces generated by the disruption predict a number of turns in the proline-rich regions and of P-turns driving the retractionof the P-turnspiral. The high helical conformations for the polyalanine regions. The Rob- tensile strength of the fiber could be derived from the high son-Garnier method predicts significant regions of P-sheet, proportion of p-sheet (oriented along the axis of the fiber). predicted secondary especially in the polyalanine segments, but the Chou-FasmanElucidation of the primary structure (and structure) of Spidroin 2, and demonstrationof the two protein subunit nature of dragline silk, has allowed disparate results Hydrophlltcity Window Slze = 7 Scale = Kyte-Doollttle 5.00 I from chemical and biophysical studiesto be incorporated I I I I I I 4.00 5 7nn ! . within a single model. ii 2.00 .E
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1.00 0.00
Acknowledgment-We thank Dr. Yoichi Kadokami for valuable discussions and technical assistance.
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REFERENCES
500
1. Lucas, F. (1964) Discovery 25, 20-26 2. Koover, J. (1987) Ecophysiology of Spiders (Nentwig, W., ed) pp. 160-188,
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II
600
FIG. 4. Predicted hydrophilicity and secondary structure of Spidroin 2. Hydrophilicity using Kyte-Doolittle evaluation of a 7amino-acid window is shown over the secondary structure predictions generated using Chou-Fasman ( C F ) or Robson-Garnier (RG) algorithms,as well asthe consensus structure (CfRg). Bothsets of predictions were generated using MacVector, version 3.5 (International Biotechnologies, Inc.) on a MacIntosh IIfx (Apple Computer, Inc.).
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K. Matsuno and R. V. Lewis, manuscript in preparation.
19324
Spider Dragline Silk
Is a Two-protein Fiber
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