High-resolution NMR characterization of a spider-silk mimetic composed of 15 tandem repeats and a CRGD motif.
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.
McLachlan
Glendon D
GD
Department of Physics and Astronomy Hunter College, City University of New York, New York, NY 10065, USA. gmclachl@hunter.cuny.edu
Slocik
Joseph
J
Mantz
Robert
R
Kaplan
David
D
Cahill
Sean
S
Girvin
Mark
M
Greenbaum
Steve
S
D000595:Amino Acid Sequence; D000818:Animals; D032701:Biomimetics; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D020005:Propanols / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics*; D047011:Silk / Q000737:chemistry* / Q000235:genetics*; D012997:Solvents / Q000378:metabolism; D013112:Spiders; D014867:Water / Q000378:metabolism; D006845:Hydrocarbons, Fluorinated
D020005:Propanols; D011994:Recombinant Proteins; D047011:Silk; D012997:Solvents; D014867:Water; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated