Spider Silk Protein Refolding Is Controlled by Changing pH

Kenney, J. M.; Knight, D.; Wise, M. J.; Vollrath, F. Amyloidogenic nature of spider ...... Jason E. Bressner , Benedetto Marelli , Guokui Qin , Lauren...
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Biomacromolecules 2004, 5, 704-710

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Spider Silk Protein Refolding Is Controlled by Changing pH Cedric Dicko,*,†,‡,§ Fritz Vollrath,†,§ and John M. Kenney‡,| Department of Zoology, Oxford University, OX1 3PS Oxford, United Kingdom, Department of Physics, East Carolina University, Greenville, North Carolina 27858, and Institute for Storage Ring Facilities and Department of Zoology, University of Aarhus, 8000 Aarhus C., Denmark Received August 19, 2003; Revised Manuscript Received February 17, 2004

Spidroins, the major silk proteins making up the spider’s dragline silk, originate in two distinct tissue layers (A and B) in the spider’s major ampullate gland. Formation of the complex thread from spidroins occurs in the lumen of the duct connected to the gland. Using pH-sensitive microelectrode probes, we showed that the spidroins traveling through the gland and duct experience a monotonic decrease in pH from 7.2 to 6.3. In addition, circular dichroism spectroscopy of material extracted from the gland showed a structural refolding concomitant with position in the gland and post-extraction changes in pH. We demonstrate that lowering the pH in vitro causes a dramatic conformational change in the protein from the A zone, converting it irreversibly from a coil to a predominantly β-sheet structure. Furthermore, amino acid analyses have indicated that there are at least two distinct, though similar, proteins secreted in the A and B zones suggesting a potential factor in the progressive acidification as well as a pH sensitivity of the folding of spidroins in the gland. Thus, we provide, for the first time, a quantitative map of the pH value and position correlated with molecular structural folding in the silk gland characterizing the crucial role that pH plays in spider silk formation. Introduction Silk is of vital importance for a spider. Indeed, orbweaving spiders can produce up to seven1,2 types of silks, each with a specific function and unique properties.2,3 The major ampullate (MA) silk that forms most of the safety line (dragline) and radial fibers of the web has received the most attention. The dragline exhibits great tensile strength combined with good extensibility4-8 thus giving it a toughness greater than most man-made fibers, including steel.9 To achieve these remarkable properties, spiders have evolved well-designed elastomeric silk proteins,10-12 as well as complex and unique spinning glands.8 The MA gland, giving rise to the spider dragline, is rather large and easily extracted making it an excellent model system to study spider silk formation. Although spiders as a group present a large diversity of silk types and functions,2 it is strongly suggested by histochemical, histological, and ultrastructural evidence that most of these silks are processed in a rather similar way.13 This covers not only silks from araneids and tetragnathidae (typical ecribellate orb weavers) but a variety of representatives of the liphistiomorph, mygalomorph, and araeonomorph suborders.1,13-19 There are also remarkable similarities8 between the silk spinning of spiders and that of silkworms (a family of insect that has independently evolved silk production). This suggests that * Author to whom correspondence should be addressed. Tel.: + 44 1865 271216. Fax: + 44 1865 281253. E-mail: [email protected]. † Oxford University. ‡ Institute for Storage Ring Facilities, University of Aarhus. § Department of Zoology, University of Aarhus. | East Carolina University.

Figure 1. pH measurement setup: (1) pH meter, (2) pH microelectrode, (3) reference electrode, (4) temperature probe, (5) temperature meter, (6) drop of Tris buffered Ringer (pH 7.4), (7) MA gland, and (8) inert rubber bottom of the Petri dish. The stereomicroscope and the electrode micromanipulators are not represented. Scale bar ) 5 mm.

a series of common characteristics and parameters are critical for the spinning process. From a gross morphological perspective, regionalization is immediately apparent in the MA gland of the Nephila species. The paired glands consist of a tubular tail portion, a sacklike midpiece (ampulla), and a fine looped duct.20-22 The ampulla has two transverse epithelial zones1,15,17 referred to as A and B (Figures 1 and 2). In the current model, the A

10.1021/bm034307c CCC: $27.50 © 2004 American Chemical Society Published on Web 04/13/2004

Silk Protein Refolding

Figure 2. Stereomicroscope image of the MA gland, with the corresponding zones (see text).

zone secretes one of the two major silk proteins (spidroin2, Sp2) to form the bulk of the fiber while the B zone secretes a protein coating (mainly spidroin1, Sp1, mixed with some Sp2).1,17,23 Although the spinning mechanism is not completely understood, it appears to involve the conversion of a highly concentrated liquid crystalline solution of the spidroins into an elastomeric solid.8,10,12,24 The process is thought to take place mainly within the S-shaped duct of the MA gland and to involve a transition in the spidroin from a predominantly random coil secondary structure24,25 into one in which β crystallites lock the molecules together into a solid fiber.8 This transition seems not unlike the formation of other “tough” protein nanofibers such as amyloids and prions as Kenney et al.24 recently showed and would, thus, seem to be a generic pathway to an extremely stable state. Although the causes of this transformation in spider silk are not yet fully understood, the silk spinning solution appears to be subjected to water resorption,26,27 a decrease in pH,15,27,28 a decrease in sodium ion concentration, an increase in potassium ion concentration,28 and a rapid extensional flow in an internal draw down taper.29 In this paper, we focus our attention on pH measurement and on changes in secondary structure of the liquid silk as it flows through the ampulla. Earlier studies indicate that the lumen in the tail and ampulla A zone portions are slightly alkaline or neutral whereas the B zone and duct are acidic.1,15,17,30 The origin of this pH gradient in the ampulla is not clear, nor has the gradient been fully quantified, though several hypotheses have been put forward. The secretion of acidic polysaccharides in the B zone and the presence of a high concentration of tyrosine residues in the A zone and tail might account for variation in pH.1,15 A high activity of phosphatases13 in epithelial cells suggests that pH may be influenced by the secretion of phosphate ions into the lumen of the gland. More recently, the amino acid composition31-34 and the partial DNA sequences of spider silk proteins35-38 indicate the presence of residues such as histidine, arginine, glutamate/ glutamine, and aspartate/asparagine that could influence the pH. Vollrath and co-workers27 presented evidence for a highly active proton pump in the tall epithelial cells that line

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the distal part of the MA duct (third limb) in Nephila edulis spiders, suggesting a progressive acidification of the silk protein during its transformation into a fiber. A strong and direct consequence of having proton pumps in the duct is that the ampulla could act as a proton sink, hence creating a pH gradient (private communication with David Knight). More recently, Knight and Vollrath28 used colored indicators for a semiquantitative in vivo determination of the pH values in Nephila senegalensis spider spinning glands, indicating a drop from pH 6.9 in the ampulla to a value of 6.3 in the distal part (third limb) of the duct. Accompanying the change in pH, the viscosity of the liquid silk increases in vitro with acidification as the result of a pH-induced gelation coupled with an increase in β-sheet content.27,39,40 A proton pump41 and similar progressive acidification have been found also in the duct of the silkworm silk gland, suggesting a generic role for acidification in natural spinning processes. We now provide the first quantitative in situ measurements of the pH at different positions along the spider’s silk secretory pathway. Our data give clear evidence for a pH gradient from the tail of the gland to the proximal part of the duct. We further present evidence from synchrotronradiation-based (SR-based) circular dichroism (CD) spectroscopy that the secondary structure of the silk protein changes as it travels slowly through the ampulla, and this change in secondary structure is triggered by a decrease in pH. This observation suggests that the spider’s regulation of the pH in the ampulla and duct is a vital factor in controlling the refolding of the major silk protein while it is being spun into a fiber. Experimental Section Spider Handling. Mature female “golden silk” spiders, N. edulis (Tetragnathidae), initially reared free-range in an environmentally controlled room, were confined to square frames (40 × 40 × 10 cm) after reaching the last instar. Every other day these spiders were fed Musca domestica flies ad libitum, and their webs were sprayed with tap water. Framing the spiders limited the spinning of silk and led to an accumulation of silk precursor protein in the ampulla of the silk gland, enabling the separation of the luminal contents into three fractions (see the following). After at least 2 weeks in the frames, the spiders’ glands were used for pH determination and to prepare samples for CD. Complementary pH measurements were conducted on free-range spiders. Gland Preparation and pH Measurement. pH microelectrodes offer a nondestructive, direct, and continuous recording method for measuring proton activity in situ at a very high spatial resolution (50 µm for our setup) without changing the activity of the protons being measured.42-44 The spider’s opisthosoma (abdomen) was cut away from the cephalothorax and immediately dissected in spider Ringer45 buffered with Tris base (100 mM) to pH 7.4. The cleaned MA gland was transferred to a Petri dish with a thin layer of inert rubber set in the bottom to protect the pH microelectrode from accidental damage. The gland was covered with a small drop of the same buffered Ringer solution. Glands were discarded if there was noticeable injury to the

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Table 1. Relative pH Values along the MA Gland

Dicko et al. Table 2. Apparent pH Values along the MA Gland pH ( SDa

∆pHa ( SD (nb) A zone start A zone middle B zone funnel duct (0.5 mm from funnel)

+0.534 ( 0.117 (7) +0.313 ( 0.056 (13) -0.173 ( 0.048 (8) -0.370 ( 0.042 (7) -0.427 ( 0.188 (7)

intermediate zonec

-0.697 ( 0.226 (22)

a ∆pH ) pH - pH x intermediate zone. pHintermediate zone - pHRinger drop.

b

Number of gland used. c ∆pH )

epithelium. The tip of the glass microelectrode (Unisens, pH50-1016, nominal diameter 45 ( 5 µm) was inserted through the epithelium of the ampulla, and the reference electrode (Unisens, REF100) was placed in the surrounding Ringer solution drop, using micromanipulators (Prior, U.K.) and a stereomicroscope (see Figure 1). These electrodes were chosen for their rapid response time, stability, and tip size. Before use, the electrodes were connected to a standard pH meter (PHM62; Radiometer, Copenhagen) and left to equilibrate for at least 1 h in a pH 4 buffer solution. The pH meter was used in difference-ofpotential mode to improve accuracy. The difference of potential was monitored until a stable reading was reached for each point of insertion. Before and after performing the difference of potential measurements, the system was calibrated (5 points calibration) with analytical purity buffers (IUPAC Standard Etalon from Radiometer Analytical, France) at room temperature (23 ( 2 °C) to obtain the final pH values. The calibration curves were temperature corrected. External factors such as electrical interferences, temperature, and vibrations were carefully monitored and controlled. For each gland, we measured the potential in the tail, ampullary A zone, intermediate zone, B zone,24 funnel, and duct at 0.5 mm from the funnel. After calculation of the corresponding apparent pH values, we calculated the pH difference value (∆pH ) pH - pHintermediate zone) using the apparent pH of the intermediate zone as a reference point (Table 1). This allowed us to pool the pH differences from different glands without the risk of introducing systematic error usually inherent in the direct measurement of absolute pH. Whether the luminal contents of the distal (downstream) end of the ampulla were yellow, or colorless as in 60% of the bicolored glands, appeared not to influence the pH of the luminal contents. Accordingly, the data from all glands were pooled. The same procedure was applied to the intermediate-zone pH, but in this case using the constant and reproducible pH of the buffered Ringer drop (∆pH ) pHintermediate zone - pHRinger drop). Finally, the apparent pH values of the different zones were calculated back from the fixed pH of the buffered drop. Twenty-two different glands were used to give statistical reliability. Hemolymphe pH Determination. Immediately after sectioning the abdomen away from the spider thorax, a pale liquid, the hemolymphe, oozed from the cut. A total of 100 µL of hemolymphe was collected and quickly spread on pHindicator strips (Universal indicator pH 0-14, Merck). The value read was 8-9 (see Table 2). Amino Acids Analysis. Samples (at least three independent samples per tissue taken from different spiders) from

Ringer buffered

dropb

7.430 ( 0.010

A zone start A zone middle intermediate zone B zone funnel duct (0.5 mm from funnel)

7.268 ( 0.353 7.046 ( 0.292 6.733 ( 0.236 6.560 ( 0.284 6.363 ( 0.278 6.306 ( 0.424

hemolymphec

8-9

a Cumulated standard deviation (SD Ringer drop, intermediate zone, and zone of interest). b From three measurements. c Measured with pH indicator strips.

the A zone, intermediate zone, and B zone were isolated after dissection (see previous text) into preweighted Eppendorf tubes and left to dry at room temperature in a desiccator. After several weeks and no further measurable changes in the sample mass, amino acid analysis was conducted using an ABI 420A derivatizer/analyzer (PE Biosystems, Warrington, U.K.) with a narrow-bore HPLC system (Applied Biosystems 130A) after hydrolysis for 24 h at 110 °C in 5.7 N hydrochloric acid. The ABI 420A utilizes precolumn derivatization46 with phenylisothiocyanate to form phenylthiocarbamyl amino acids. Data handling was performed using Dionex Chromeleon software (version 6.40 from Dionex U.K., Ltd., Macclesfield, U.K.). SR-Based CD. CD is useful for monitoring the effects of various conditions on protein secondary structure. The extreme brightness and wide spectral range47 of SR-based CD spectrometers allow data to be collected with high spatial resolution, with low noise background, and, most importantly, under near in vivo conditions, making it valuable for studying concentrated, light-scattering protein solutions, such as those of spider silks.48,49 In addition, SR CD permits observations down to 130-nm wavelengths, greatly extending the range of conventional CD measurements (190-300 nm), thus resulting in better fold recognition or tertiary structure determination.50-52 Spiders were dissected as described previously, and the MA gland was collected in 10 mM phosphate buffer at pH 7. The epithelium was gently stripped off. In most glands (60%), there was a clear distinction between the contents in the A zone and the B zone (see Figure 2), enabling material from the lumen to be divided into three fractions by two transverse cuts: (1) a homogeneous, yellow, somewhat translucent A zone secretion in the proximal (upstream) part of the ampulla, (2) a clear, colorless fraction derived predominantly from the B zone, and (3) an intermediate fraction (pale yellow) containing predominantly A-zone secretion with a little B-zone secretion taken from the lumen of the gland in the proximal part of the B zone (see Figure 2). For shorthand, fraction 1 is hereafter referred to as A-zone material and fraction 2 as B-zone material. These fractions were blotted dry with lens paper and collected in preweighed Eppendorf tubes. After reweighing, the material was dissolved overnight at 5 °C in 10 mM phosphate buffer at both pH 7.2 and 6.35, to give a 20% solution (blotted w/v). Care was taken to avoid shearing the protein solutions at all times, because this material is very sensitive to strain, particularly

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Silk Protein Refolding Table 3. Amino Acid Composition along the Ampullate Gland amino acid

A zone composition (%) ( SDa

intermediate zone composition (%) ( SD

B zone composition (%) ( SD

Asxb Glxc Ser Gly Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys

1.15 ( 0.15 13.63 ( 1.28 2.03 ( 0.11 32.78 ( 2.37 1 ( 0.09 0.56 ( 0.07 26.83 ( 1.34 12.71 ( 0.85 2.66 ( 0.42 1 ( 0.18 0.16 ( 0.03 0.08 ( 0.03 0.6 ( 0.09 2.66 ( 0.3 0.37 ( 0.07 0.41 ( 0.06

1.16 ( 0.19 13.8 ( 1.72 1.84 ( 0 41.23 ( 0.42 1.22 ( 0.22 0.62 ( 0 16.56 ( 2.69 13.12 ( 0.62 3.26 ( 0.64 1.13 ( 0.13 0.17 ( 0.03 0.03 ( 0.007 0.72 ( 0.09 2.9 ( 0.24 0.22 ( 0.13 0.51 ( 0.08

0.99 ( 0.06 11.5 ( 0.56 2.53 ( 0.18 47 ( 0.85 1.15 ( 0.42 0.49 ( 0.09 14.2 ( 0.49 9.99 ( 0.99 3.13 ( 0.02 0.99 ( 0.06 0.23 ( 0.02 0.03 ( 0.04 1 ( 0.54 3.27 ( 0.93 0.45 ( 0.07 0.38 ( 0.15

a

Standard deviation. b Asx: Asp and Asn. c Glx: Glu and Gln.

when concentrated.40 Samples were transferred to a 10-µm path length Suprasil quartz open cell (Hellma 124-QS). Spectra were collected at 20 °C on the SR-based CD facility at ISA, Aarhus, Denmark. The control voltage that determines the high-tension (HT) dynode voltage of the photomultiplier tube is recorded with each CD spectrum to indicate the reliability of each CD spectrum. In all our measurements, the control voltage was -3 V (corresponding to a +600 V HT) for a completely transmitting sample and +7 V (+1600 V HT) for a totally absorbing sample. Results Figure 2 shows the structure of the MA gland in N. edulis and the different zone colorations. The pH microelectrode measurements are summarized in Tables 1 and 2. The first part of Table 1 shows the differences in the pHs between all the identified zones and the intermediate zone, and the second part shows the difference in pH between the intermediate zone and the buffered Ringer drop surrounding the gland. The pH values of all zones calculated by reference to the pH of the buffered Ringer drop are presented in Table 2 (see experimental part for details). A plot of the mean pH value in each zone against the approximate position along the production pathway (data not shown) showed that the pH decreased monotonically from pH 7.2 in the tail of the ampulla to about pH 6.3 at the start of the duct. The amino acid composition (Table 3) of the A-zone material was very similar to that of Nephila claVipes spiders33,34 with a predominance of alanine (Ala) and glycine (Gly) residues and a marked absence of histidine residues. However, along the glands there was no change in composition except for two amino acids: alanine and glycine. The gland content of alanine showed a significant decrease with the protein flow whereas the content of glycine showed a significant increase, suggesting an addition of a glycine-rich compound in the B zone. Note the presence of glutamate/

Figure 3. CD spectra of A-zone and B-zone luminal contents at a concentration of 20% (blotted w/v) in 10 mM phosphate buffer. CD and HT: (0) A zone, pH 7.2; (O) A zone, pH 6.35; (∆) B zone, pH 7.2; and (∇) B zone, pH 6.35.

glutamine and aspartic acid/asparagine that could contribute to the final pH of the silk secretions. CD spectra were obtained for usually yellow A-zone material and for the usually colorless B-zone material buffered to the pH values of 7.2 and 6.35, the pH values of the luminal contents at the start of the A zone and end of the B zone, respectively (Figure 3). These values were selected to study the structure of the protein at the pH it had in the lumen and to follow how the structure might change as the protein flowed through the ampulla in vivo. The CD signature of the A-zone material at pH 7.2 showed a negative band at 199 nm traditionally indicating a predominantly “random coil” or open conformation and a negative plateau centered at 217 nm attributed to β-sheet structure.24 When subjected to acidification (pH 6.35), the A-zone material underwent a slow (overnight) structural transition toward a β-rich type of structure (see Figure 3). The intermediate zone showed no significant difference from A-zone spectra (data not shown). In contrast, the B zones at pH 7.2 and pH 6.35 and the A zone at pH 6.35 presented a similar β-sheet-rich spectrum24 with a positive peak at around 188 nm, a positive shoulder at around 198 nm, and a negative band at 217220 nm. The shoulder at 198 nm and the remaining positive band at 188 nm suggested a mixed structure, intermediate between the predominantly random coil A zone and the β-sheet-rich structure24 of the B-zone material. Discussion Our pH measurements extend, in both the resolution of the pH value and the spatial position, the semiquantitative and two-position evaluation of the pH in the ampulla and duct obtained by Knight and Vollrath28 giving us a much more complete as well as quantitative picture of the pH variation within the ampulla under nearly in vivo conditions. Thus, the question is posed: what establishes this pH gradient? Histochemical evidence15,19 suggests that several different classes of macromolecule are passed into the lumen of the A and B zones of the MA glands in a pattern that is more or less consistent across different suborders of spider. The A zone appears to secrete a protein with an alkaline reaction while the B zone secretes protein together with an acidic polysaccharide.15 The amino acid analysis showed that glutamine/glutamate and to a lesser extent aspartic acid/

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Figure 4. Flow chart summarizing the correlation between pH and the structure along the spinning pathway (the shape of the B-zone box represents the narrowing of the B zone in the gland).

asparagine residues could contribute via changes in protein folding to the establishment of the pH gradient we observed. Though the presence of proton pumps in the third limb of the duct creating a proton sink upstream in the ampulla could provide a strong enough electrochemical force to establish a pH gradient (personal communication with David Knight). One independent observation worth noting is the coloration of the silk and the large variability in the “golden sheen”. Only the Nephila species, also referred to as golden orb spiders, present this gold-colored dragline silk, which is already reflected in the color of the glands. An early study53 suggested that the pigmentation resulted from a mixture of benzoquinone, naphthoquinone, and xanthurenic acid and their probable role as antibacterial agents. For us, interested in the spinning process, is the implication of coloration changes from yellow to colorless upon acidification.53 To this end, a relatively strong acid character of the pigmentation (unpublished observations under study) might suggest a more active role of the yellow coloration in controlling the pH in the gland. In the perspective of the spinning process, it is now accepted that the formation of fibers correlates with an increase in β-sheet structure content.24,54-58 Further understanding of this correlation was described in recent rheological investigations,40 demonstrating a pH-dependent shear sensitivity of the liquid silk in N. senegalensis spiders giving an optimal value for maximum shear sensitivity at pH 6.4 to form fibers. In our study, the closest value to this was pH 6.3. This points to the region between the B zone and the funnel as the position in the gland where the liquid silk would be the most shear sensitive and would undergo some structural rearrangement.12 In addition, it appeared that neutrality or slight alkalinity as measured in zone A corresponded to the pH for optimal storage. The results from CD showed that the spectroscopic signature of the A zone at pH 7.2 is similar to the spectrum for the A zone in Tris buffered (pH 7.4) Ringer solution reported by Kenney et al.24 and interpreted as predominantly random coil with some β sheets and turns structures. In an earlier study, Hijirida et al.25 reported a CD spectrum of the full gland content different from the A zone we found but similar to one of the intermediates found by Kenney et al.24 in a temperature melt experiment, stressing the difficulty to

work with the native silk proteins. A further interpretation of the spectra in terms of secondary structures content was difficult, without the knowledge of the sample concentration and a bias toward globular protein secondary structures in the database used to analyze the CD data. However, NMR studies by Hijirida et al.25 and Kummerlen et al.59 suggested the presence of noncanonical secondary structures such as 31 helices in the liquid silk. Threefold-type structures were further confirmed in the secondary structure elements predicted from the gene sequences of Sp1 and Sp2.36,37,60 Films prepared from the native MA gland25,61 showed using Fourier transform infrared spectroscopy predominantly some random coil, R helices and turns with little amounts of β sheets, suggesting a structure similar to the silk I found in silkworm silk. Although suggestive of the presence of conformations other than R helices and β sheets, our CD data were not compelling enough to identify other conformations. But a clear change in conformation was observed as a function of the position in the gland and by decreasing the pH. The structural rearrangement we observed was shown to be associated with a different amino acid composition, suggesting a nonuniform distribution of the silk proteins. A qualitative comparison of the cDNA dominant repeats37,60 of Sp1 and Sp2 showed that Sp2 is proline-rich, whereas in Sp1 proline residues are absent, suggesting from the amino acid composition (Table 3) that Sp2 is secreted already from the A zone. This conclusion was reached by considering, first, that the proteins flow from the tail to the duct and that there are at least two types of epithelial cells along the ampulla and, second, that the amino acid composition remained approximately constant for all residues except alanine, glycine, and, to a lesser extent, proline, suggesting the addition of one or more proteins as depicted in Figure 4. The marked decrease in alanine content could only be explained by differences in protein composition in the A and B zones. The B zone would secrete one or more proteins rich in glycine residues not found in the A zone secretion. The ratio of alanine to glycine in each part matched closely the observed ratio drawn from the cDNA. Thus, in the A/intermediate zones predominantly Sp2 is secreted, and in the B zone a mixture of Sp1 and Sp2 is secreted with the

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Silk Protein Refolding

two proteins arranged so that as Sp2 flows, Sp1 will be added as a coat19 (Figure 4). Observations using specific antibodies to Sp1 and Sp2 provide some evidence for this organization in silk fibers.62 Though the A and B zones showed different spectral signatures and different compositions, no particular inference could be made about the contribution from the B zone material to the structural transition in the silk proteins. Nevertheless, the observed change in pH within the gland suggested a casual relationship between the structural rearrangement from a predominantly random coil structure to the predominantly β-sheet-like structure in the gland extracts and a change to a more acidic pH in the gland (Figure 4). This is supported by the observation that the acidification in vitro of the A zone material resulted in a change in structure toward an increase in the β-sheet content. Furthermore, it is worth noting that these changes are likely to be irreversible, as suggested by the observations on the effect of heating on silk proteins.24 Finally, these results, therefore, suggested that the regulation of pH appears to be important in driving the fiber spinning process, particularly the initiation of fiber formation. Conclusion We have characterized under nearly in vivo conditions the pH gradient and the associated CD structure of the material in the ampulla and the start of the duct. The direct pH measurements together with the CD spectrographic analyses imply that the spinning of the liquid silk involves a progressive acidification concomitant with protein structure refolding. The site-specific amino acid analysis evidence for at least two types of similar, though distinguishable, major proteins in the ampulla is indicative of a complex spinning process in which pH, though critical, is not the only factor. In any case, the control of spidroin folding associated with the variation of pH provides strong evidence for the role of pH in storing, transporting, and converting the spider silk protein. Although other factors such as the concentration, co-aggregation agents, time, mechanical stress, and composition need to be considered to fully understand the transition from liquid silk to solid fiber, our work demonstrates that the structure of the proteins in the gland responsible for dragline silk fiber formation correlates with the pH in the gland and that acidifying the pH can drive the structural refolding that apparently leads to fiber formation. Acknowledgment. We thank the British Biological and Engineering Research Councils (BBSRC, EPSRC) and the Danish Natural Sciences Research Council (SNF) for financial support and the Institute for Synchrotron Radiation, ISA, Aarhus, Denmark, for use of their CD facility. We are grateful to Dr. David Knight and Dr. Ann Terry for reading the manuscript and helpful discussion, Dr. Søren V. Hoffman for his help and assistance with the CD beamline at ISA, Else Rasmussen for technical support, and Dr. Anthony Willis for the amino acids analysis. References and Notes (1) Kovoor, J. La soie et les glandes sericigenes des arachnides. Annee Biol. 1977, 16, 97-171.

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