Structure of disordered reflectin derived polypeptide Ref(2C)₄ assembly by solid state NMR

MD Imran Khan¹, Tyrone Thames¹, Ivan Hung², Zhehong Gan², and Bo Chen¹

1. University of Central Florida, Orlando, FL
2. National High Magnetic Field Laboratory, Tallahassee, FL

Cephalopods can shift their dazzling body color instantaneously owing to the self-assembly formed by a structural protein called reflectin.¹ However, its structural basis remains unclear at the molecular level. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy analyses showed that reflectin assumes largely disordered structure in its assembly, not suitable for high-resolution characterization techniques such as X-ray crystallography or cryoEM. Ref(2C)₄ is a polypeptide designed by linking four repeats of the conserved sequence DPRYYDYYGRFNDYDRYYGRSMF in E. Scolopes reflectin 1b.² Its assembly preserves the color shifting function with superb proton conductivity.³ Hence, it is a suitable template to probe the fascinating structure-functional correlation of reflectin.

In this work, we present the first structural model of Ref(2C)₄ aassembly determined by ssNMR. Its NMR spectra display broad resonances (1.2 to 3 ppm), consistent with prior CD and FTIR analyses of the assembly formed by full length reflectin. Accurate residue-specific assignments were determined by ¹³C, ¹⁵N uniformly labeled and selective-residue labeled samples. Subsequently, consistent sequential assignments were obtained with our recently invented auto-assignment program ASAP.⁴  Based on NMR chemical shift assignments, TALOS-N predictions revealed that Ref(2C)₄ adopts short alpha-helices sparsely spaced by random coils. In addition, assembly interface was resolved by an assembly sample absorbing MnCl₂ to quench resonances of solvent exposed residues. Tertiary contacts were identified between Tyr and Arg with mixed labeled samples, with extensive contacts between their side-chains that contribute high proton conductivity.

References

1.Crookes, W. J.; Ding, L.-L; Huang, Q. L.; Kimbell, J. R.; Horwitz, J.; McFall-Ngai, M. J.; Reflectins: The Unusual Proteins of Squid reflective Tissues. Science 2004, 303, 235-238.

2.Dennis, P. B.; Singh, K. M.; Vasudev, M. C.; Naik, R. R.; Crookes-Goodson, W. J. Research Update: A Minimal Region of Squid Reflectin for Vapor-Induced Light Scattering. APL Mater. 2017, 5, 120701.

3.Xu, Chengyi; Kandel, N.; Qiao, X.; Khan, Md. Imran; Pratakshya, P.; Tolouei, N. E., Chen, B. and Gorodetsky, A. Al. Long-Range Proton Transport in Films from a Reflectin-Derived Polypeptide. ACS Appl. Mater. Interfaces. 2021, 13, 20938-20946.

4. ASAP: an automatic sequential assignment program for congested multidimensional solid state NMR spectra. JMR. 2024, 361, 107664

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