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A highly destabilizing mutation, G37A, of the bovine pancreatic trypsin inhibitor retains the average native conformation but greatly increases local flexibility.

Battiste JL, Li R, Woodward C
Biochemistry 41 2237-45 (2002)
Cited: 15 times
EuropePMC logo PMID: 11841215

Abstract

A point mutation, G37A, on the surface of bovine pancreatic trypsin inhibitor (BPTI) destabilizes the protein by approximately 5 kcal/mol, which is very high for addition of one methyl group. In wild-type (WT) BPTI, Gly 37 HN is in an unusual NH-aromatic-NH network of interactions with the ring of Tyr 35 and the side chain HN of Asn 44. G37A was designed to disrupt this interaction, since the phi and psi backbone angles of G37 are not favorable for an amino acid containing a beta-carbon. Investigations of the structure and dynamics by NMR methods show that G37A retains the average WT structure. The NH-aromatic-NH interactions remain intact, as indicated by NOEs and the large upfield ring current shift (approximately 4 ppm) of A37 HN. The NMR structure, confirmed by molecular modeling calculations, requires phi and psi backbone angles that are highly destabilizing when alanine is in position 37. Although the average structure is essentially unchanged, the dynamics are altered dramatically. Many residues in the region of the mutation have increased flexibility, as probed by aromatic ring flip rates and native state hydrogen exchange. We conclude that a large fraction of the destabilization arises from maintaining A37 in a high-energy conformation. This suggests that disruption of the NH-aromatic-NH network is energetically very costly, and may involve other cooperatively linked interactions. The results illustrate the importance of the Gly-Gly sequence at positions 36 and 37 and the 37 HN-35 aromatic interaction to the stability, folding, and dynamics of the BPTI.

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  2. Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein. Alcaraz LA, del Alamo M, Barrera FN, Mateu MG, Neira JL. Biophys J 93 1264-1276 (2007)
  3. How amide hydrogens exchange in native proteins. Persson F, Halle B. Proc Natl Acad Sci U S A 112 10383-10388 (2015)
  4. Wiggle-predicting functionally flexible regions from primary sequence. Gu J, Gribskov M, Bourne PE. PLoS Comput Biol 2 e90 (2006)
  5. Ligand-induced conformational heterogeneity of cytochrome P450 CYP119 identified by 2D NMR spectroscopy with the unnatural amino acid (13)C-p-methoxyphenylalanine. Lampe JN, Floor SN, Gross JD, Nishida CR, Jiang Y, Trnka MJ, Ortiz de Montellano PR. J Am Chem Soc 130 16168-16169 (2008)
  6. Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged. Verma D, Jacobs DJ, Livesay DR. PLoS Comput Biol 8 e1002409 (2012)
  7. Light chain somatic mutations change thermodynamics of binding and water coordination in the HyHEL-10 family of antibodies. Acchione M, Lipschultz CA, DeSantis ME, Shanmuganathan A, Li M, Wlodawer A, Tarasov S, Smith-Gill SJ. Mol Immunol 47 457-464 (2009)
  8. The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function. Wilson KA, Bär S, Maerz AL, Alizon M, Poumbourios P. J Virol 79 4533-4539 (2005)
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  10. The role of site-directed point mutations in protein misfolding. Baruah A, Biswas P. Phys Chem Chem Phys 16 13964-13973 (2014)
  11. NMR structures of two variants of bovine pancreatic trypsin inhibitor (BPTI) reveal unexpected influence of mutations on protein structure and stability. Cierpicki T, Otlewski J. J Mol Biol 321 647-658 (2002)
  12. Experimentally assessing molecular dynamics sampling of the protein native state conformational distribution. Hernández G, Anderson JS, LeMaster DM. Biophys Chem 163-164 21-34 (2012)
  13. Interpreting Hydrogen-Deuterium Exchange Experiments with Molecular Simulations: Tutorials and Applications of the HDXer Ensemble Reweighting Software [Article v1.0]. Lee PS, Bradshaw RT, Marinelli F, Kihn K, Smith A, Wintrode PL, Deredge DJ, Faraldo-Gómez JD, Forrest LR. Living J Comput Mol Sci 3 1521 (2021)


Related citations provided by authors (1)

  1. Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding.. Li R, Battiste JL, Woodward C Biochemistry 41 2246-53 (2002)

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