Protein Engineering of FN3 published

Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain

Benjamin T. Porebski, Adrian A. Nickson, David E. Hoke, Morag R. Hunter, Liguang Zhu, Sheena McGowan, Geoffrey I. Webb and Ashley M. Buckle

Protein Engineering, Design and Selection (2015) 28 (3): 67-78. doi: 10.1093/protein/gzv002

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Consensus protein design is a rapid and reliable technique for the improvement of protein stability, which relies on the use of homologous protein sequences. To enhance the stability of a fibronectin type III (FN3) domain, consensus design was employed using an alignment of 2123 sequences. The resulting FN3 domain, FN3con, has unprecedented stability, with a melting temperature >100°C, a ΔGD−N of 15.5 kcal mol−1 and a greatly reduced unfolding rate compared with wild-type. To determine the underlying molecular basis for stability, an X-ray crystal structure of FN3con was determined to 2.0 Å and compared with other FN3 domains of varying stabilities. The structure of FN3con reveals significantly increased salt bridge interactions that are cooperatively networked, and a highly optimized hydrophobic core. Molecular dynamics simulations of FN3con and comparison structures show the cooperative power of electrostatic and hydrophobic networks in improving FN3con stability. Taken together, our data reveal that FN3con stability does not result from a single mechanism, but rather the combination of several features and the removal of non-conserved, unfavorable interactions. The large number of sequences employed in this study has most likely enhanced the robustness of the consensus design, which is now possible due to the increased sequence availability in the post-genomic era. These studies increase our knowledge of the molecular mechanisms that govern stability and demonstrate the rising potential for enhancing stability via the consensus method.

Review Published on T-cell Receptor Dynamics

Understanding the structural dynamics of TCR-pMHC complex interactions.
Kass I, Buckle AM, Borg NA.

Trends Immunol. 2014 Nov 11;35(12):604-612. doi: 10.1016/ [Epub ahead of print]


Exponential growth in simulation complexity over time. Growth is driven by the doubling of computer power every 24 months coupled with algorithmic advances. The complexity of the simulation is represented by the product of the timescale and system size, on the y-axis. Filled circles represent published molecular dynamics (MD) studies.

Dynamics plays an important but underappreciated role in the interaction between the T cell receptor (TCR) and peptide-bound major histocompatibility complex (pMHC). Crystallographic studies have provided key molecular insights into this interaction; however, due to inherent features of the structural approach, the image of TCR-pMHC interactions that has emerged is a static one. In this review, we discuss how molecular dynamics (MD) simulations can complement and extend current experimental methods aimed at examining TCR-pMHC dynamics. We review the insights obtained from studies that leverage MD approaches, and propose that an integrative strategy that harnesses both MD simulations and structural and biophysical methods will provide new inroads into understanding the transitory and dynamic molecular events that dictate TCR signaling and T cell activation.

New papers published

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease.

Hargreaves CE, Grasso M, Hampe CS, Stenkova A, Atkinson S, Joshua GW, Wren BW, Buckle AM, Dunn-Walters D, Banga JP.

Molecular determinants of the substrate specificity of the complement initiating protease, C1r.

Wijeyewickrema LC, Yongqing T, Tran TP, Thompson PE, Viljoen JE, Coetzer TH, Duncan RC, Kass I, Buckle AM, Pike RN.

J Biol Chem. 2013 Apr 15. [Epub ahead of print]

Mechanism-based selection of a potent kallikrein-related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI-1.

de Veer SJ, Ukolova SS, Munro CA, Swedberg JE, Buckle AM, Harris JM.

Biopolymers. 2013 Mar 11. doi: 10.1002/peps.22231. [Epub ahead of print]

Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates.

Langendorf CG, Tuck KL, Key TL, Fenalti G, Pike RN, Rosado CJ, Wong AS, Buckle AM, Law RH, Whisstock JC.

Biosci Rep. 2013 Jan 11;33(1):137-44. doi: 10.1042/BSR20120111.

Two new papers published


Schmidberger JS,  Bate MA, Reboul CF, Androulakis SG, Phan JMN, Whisstock JC, Goscinski WJ, Abramson A, and Buckle AM (2010) MrGrid: A Portable Grid Based Molecular Replacement Pipeline. PLoS One. Apr 6;5(4):e10049. PubMed link

Apple University Consortium (AUC) in Australia wrote an article on our grid computing in their newsletter Wheels of the Mind (PDF)


Konagurthu AS, Reboul CF, Schmidberger JS,  Irving, JA, Lesk AM, Stuckey PJ, Whisstock JC, and Buckle AM (2010) MUSTANG-MR Structural Sieving Server: Applications in Protein Structural Analysis and Crystallography. PLoS One. Apr 6;5(4):e10048.  PubMed link

Four new papers published:

Swedberg JE, Nigon LV, Reid JC, de Veer SJ, Walpole CM, Stephens CR, Walsh TP, Takayama TK, Hooper JD, Clements JA, Buckle AM & Harris, JM.  (2009) Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4.  Chemistry & Biology, 16, 633-43. PubMed Link

Fischer K,Langendorf  CG, Irving  JA, Reynolds S, Willis C, Beckham S, Law RHP, Yang S, Bashtannyk-Puhalovich TA, McGowan S, Whisstock JC, PikeRN, KempDJ, and Buckle AM  (2009) Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues. J. Mol. Biol. 390(4):635-45. PubMed Link

Fenalti G and Buckle AM (2010) Structural Biology of the GAD Autoantigen. Autoimmunity Reviews 9(3):148-52. Epub 2009 May 22. PubMed Link

Arafat Y, Fenalti G, Whisstock JC, Mackay IR, Garcia de la Banda M, Rowley MJ and Buckle AM (2009) Structural Determinants of GAD Antigenicity. Molecular Immunology, 47(2-3):493-505. PubMed Link

TARDIS article on P212121

The crystallography blog P212121 have posted an article on TARDIS  entitled “TARDIS for the Storage of X-ray Diffraction Images“.

This comes as a follow-up to their recent poll “Do we need an X-ray Diffraction Image Data Bank?“.

The poll is still open, but currently 81% of P212121 readers are voting for that the crystallography community does need an open, freely available diffraction image data bank.

Be sure to head over to P212121 and have your say.