High-resolution NMR characterization of a spider-silk mimetic composed of 15 tandem repeats and a CRGD motif

Abstract

Multidimensional solution NMR spectroscopic techniques have been used to obtain atomic level information about a recombinant spider silk construct in hexafluoro-isopropanol (HFIP). The synthetic 49 kDa silk-like protein mimics authentic silk from Nephila clavipes, with the inclusion of an extracellular matrix recognition motif. 2D (1)H-(15)N HSQC NMR spectroscopy reveals 33 cross peaks, which were assigned to amino acid residues in the semicrystalline repeat units. Signals from the amorphous segments in the primary sequence were weak and broad, suggesting that this region is highly dynamic and undergoing conformational exchange. An analysis of the deviations of the (13)C(alpha), (13)C(beta), and (13)CO chemical shifts relative to the expected random coil values reveals two highly alpha-helical regions from amino acid 12-19 and 26-32, which comprise the polyalanine track and a GGLGSQ sequence. This finding is further supported by phi-value analysis and sequential and medium-range NOE interactions. Pulsed field gradient NMR measurements indicate that the topology of the silk mimetic in HFIP is nonglobular. Moreover, the 3D (15)N-NOESY HSQC spectrum exhibits few long-range NOEs. Similar spectral features have been observed for repeat modules in other polypeptides and are characteristic of an elongated conformation. The results provide a residue-specific description of a silk sequence in nonaqueous solution and may be insightful for understanding the fold and topology of highly concentrated, stable silk before spinning. Additionally, the insights obtained may find application in future design and large-scale production and storage of synthetic silks in organic solvents.

Figure 1

The primary sequence of the recombinant 49-kDa CRGD protein. The bold typed residues in brackets comprise the 33-amino acid repeat and the RGD motif is located adjacent to the N-terminus and C-terminus.

Figure 2

2D ¹H-¹⁵N HSQC spectrum of the 49 kDa CRGD-15 repeat protein in 100% HFIP and 25°C. The 33 cross peaks, which are visible in the 2D-HSQC spectrum were assigned to the 33-amino acid repeats. The cross peaks are labeled according to the three-letter convention for amino acids and the residues labeled 1–33 correspond to the repeating residues starting at T55 as seen in Figure 1. Resonances representing the nonrepeating N and C-terminal sequences, which flank the 33 repeat-region, are not visible.

Figure 3

Strips of 3D HNCA/CBCA(CO)NH data sets utilized for sequential assignment of the backbone C_α resonances. Panels A and B displays the sequential connectivity for residues 10 to 1 and 19 to 10, respectively, of the 33-amino acid repeat. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Deviations from random coil values of the (A) C_α, (B) C_β, and (C) CO chemical shifts. Well populated helical segments are positive with deviations greater than 1 ppm from coil values for Cα and CO chemical shifts. The inverse relationship is observed for the Cβ chemical shifts. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5

Summary of sequential and medium range backbone and sidechain NOE interactions. The sequential (residues i to i+1) and medium range (residues i to i+3 and i to i+4) NOEs are represented by the lines below the primary sequence and the line thickness is an indication of the relative intensities of the NOEs.

Figure 6

Pulse field gradient translational diffusion measurement of CRGD in 100% HFIP and 25°C. Shown is the attenuation of the signal intensity as a function of the square of the gradient strength for the 1.25 mM sample and known globular proteins. The arrows point to the fast and slow components of the decay curve. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7

3D ¹⁵N-NOESY-HSQC strip-plot of the 33-amino acid repeat region from residue 24–33. The strips are obtained from the ¹⁵N plane of the NOESY-HSQC and strong intraresidue and sequential αH_i to NH_i+1 NOEs are shown with dotted lines. The weaker amide proton to side-chain NOEs, indicative of long-range interactions are labeled.

Figure 8

Circular dichroism spectra of CRGD in HFIP at 25°C. The effect of increasing H₂O content on the secondary structure of the CRGD peptide at high concentration is shown. ♦ 80% HFIP/20% H₂O, ▪ 50% HFIP/50% H₂O, ▴ 40% HFIP/60% H₂O, • 30% HFIP/70% H₂O, * 20% HFIP/80% H₂O, x 10% HFIP/90% H₂O. Each spectrum was collected at a constant protein concentration of 4 mg/mL.