Molecular Modeling Studies on Silk Peptides - ACS Publications

Chapter 23 Molecular Modeling Studies on Silk Peptides Stephen A. Fossey and David Kaplan

Downloaded by CORNELL UNIV on July 26, 2016 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch023

Biotechnology Division, Natick Research, Development, and Engineering Center, U.S. Army, Natick, MA 01760-5020

A model for the meta-stable state, silk I, of B. mori silk fibroin based on conformational energy calculations on representative silk peptides is discussed(1). In addition the model and other models for the silk I form are compared with the available experimental data from X-ray and electron diffraction and NMR and IR spectroscopy. Silk-like proteins are of interest for a number of applications. Among these are high performance fibers (2), enzyme immobilizing substrates (3) and polymers with highly controlled crystal morphologies (4). The last area capitalizes on the ability of genetic means of production to precisely control the primary sequence of the polymer and the self-assembly properties of proteins. Silk fibroin is a block copolypeptide WITH crystalline domains characterized by a Gly-X repeat where X is Ala or Ser, and of less crystalline domains in which the Gly-X repeat is common but which contain a higher fraction of residues with large side chains(5). The crystalline domains of Bombyx mori can exist in one of two different morphologies. The more stable one is known as silk Π (6,7). A detailed structural model for silk Π was first proposed by Marsh et at. (8) and refined by Fraser et al. (9,10) Its basic feature, a packing of antiparallel, β-pleated sheets, is generally accepted. More recently Takahashi et al. (11), based on the intensity of X-ray diffraction reflections have proposed that most of the β-sheets in silkfibroinare antipolar rather than polar as proposed by Marsh et al. (see Methods for a description of polar and anti-polar). Despite a long history of interest in the less stable silk I form it has remained poorly understood. Attempts to induce orientation of the silk I form for studies by X-ray or electron diffraction or solid state NMR cause the silk I form to convert to the more stable silk Π form The silk I form can be obtained by letting the contents of the silk gland dry undisturbed (9). If the contents of die gland are mechanically sheared or treated in a number of other ways, the silk Π form is obtained (9,12). The sequence of the repeating unit of the crystalline fraction of B. mori silkfibroinis (13) G-A-G-A-G-S-G-A-A-G[S-G-(A-G) ] Y n

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This chapter not subject to U.S. copyright Published 1994 American Chemical Society Kaplan et al.; Silk Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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where η is usually 2 and averages to 2. Poly(Iu-Ala Gly) has often been used as a model for the crystalline fraction of B. mori silkfibroinsince the two crystalline forms of poly(L-Ala-Gly) have been shown to be isomorphous with the two crystalline forms of silk. Experimental studies of silk I have consisted primarily of powder X-ray diffraction (5,7,14,15), electron diffraction, (14) and solid state ^ C - N M R spectroscopy (12,1418). From these studies, it has not been possible to define a unique structure for silk I. Attempts to determine a structure were based on model building and comparison of the predictions of these models with experimental data. A number of models have been proposed, including the "crankshaft" model (14) and one based on a loose fourfold helix (79). Conformational energy calculations on representative model polypeptides have been applied to the structure of collagen(20,27), poly(glycine) (22,23), poly(alanine)(24), and copolymers of alanine and glycine (24). These efforts have proceeded from model building to comparison with experimental data, but have had some success in elucidating more structural detail than available from experimental results. Methods Fossey et al. (7) have used conformational energy calculations to investigate the structure of silk-like peptides. The calculations were carried out by using the ECEPP/2 (Empirical Conformational Energy Program for Peptides) algorithm(25-27). The details of the calculations have been presented elsewhere, therefore only an overview of the approach is given here. Starting Conformations. In all cases, β-sheets (regions Ε and C of Figure 1.) consisting offivestrands of six residues, each of alternating alanine and glycine, were studied. The work of Chou et al. (28) shows that there was little difference in the dihedral angles of sheets composed of either four or eight residues or of two or three strands. The primary effect of increased chain length observed was a slight decrease in the twist generally associated with β-sheets in globular proteins. A six residue peptide was chosen to anticipate the inclusion of serine, which in B. mori silk crystalline domains appears once in each six residues. Because of the two fold symmetry of βstrands the sidechains of consecutive residues along the strands project from opposite sides of the β-sheet (29). Therefore, in a poly (L-Ala-Gly) strand, all Ala side chains point to the same side. There are then two ways in which adjacent strands can be related. The methyl side chains of the alanine residues in these strands can point to either the same side or to opposite sides of the β-sheet. The sheets in which methyl groups of adjacent strands point to opposite sides of the sheet are referred to as "out-ofregister". Those in which all methyl groups point to the same side of the sheet are referred to as "in-register." Takahashi et al. (77) have used the nomenclature polar (Figure 2a) and anti-polar (Figure 2b) to refer to in-register and out-of-register respectively, this more common usage will be adopted for the remainder of the paper. Both parallel and antiparallel as well as polar and anti-polar β-sheets were considered. In addition, various packing arrangements for the sheets were considered in the calculations. This approach constitutes a search of conformational space for sheet structures. Determination of the Unit Cell. To be consistent with prior studies on silk the b axis is taken along the strand, the a axis is perpendicular to the strand in the plane of the sheet, and the c axis is in the direction between sheets. The dimensions of a unit cell correspond to the distance between repeating units in each direction, two residues (Ala-

Kaplan et al.; Silk Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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SILK POLYMERS:

MATERIALS

SCIENCE A N D

B I O T E C H N O L O G Y

180

• Aid (silk I) 135 H * 90

•

(A

D

S

G*

A *

Gly(silk

S 45 km u>

left handed alpha helix B*

0

Θ-

-45

i G

-90

A right ι

D*

· handed alpha helix _ ι ι ι C *

-135 F* -180 -180

-135

-90

-45 φ

Figure

1.

φ-φ

0

45

90

135

180

(degrees)

M a p (adapted

f r o m ref.

31)


Molecular Modeling Studies on Silk Peptides


23. FOSSEY & KAPLAN

Figure 2. Silk II (a) polar, (b) anti-polar.


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SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

Gly) along the b axis and two adjacent chains in the sheet along the a axis. In the direction of sheet packing (c axis), the repeating unit contains two adjacent sheets for most structures discussed here, with one exception: in the structure referred to later as monoclinic, the repeating unit along the c axis contains only one sheet instead of two, because strands in adjacent sheets are identical.


Results Conformational Energies. All energy-minimized single-sheet structures had a right-handed twist, as found previously (28,30). For each of the four types of sheets, energy minima were found in the C and Εregions(37) of the φ-φ map. Figure 1 shows the regions of the φ-φ map as well as location of the dihedral angles for the FD conformation. All of those minima that were located in the Cregionhad dihedral angles of approximately (φ,

Molecular Modeling Studies on Silk Peptides - ACS Publications

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