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Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions.
J Mol Biol. 2003 Nov 21; 334(2):293-307.JM

Abstract

The folding pathways of four mutants in which bulky hydrophobic residues in the B helix of apomyoglobin (ApoMb) are replaced by alanine (I28A, L29A, I30A, and L32A) have been analyzed using equilibrium and kinetic methods employing NMR, CD, fluorescence and mass spectrometry. Hydrogen exchange pulse-labeling followed by mass spectrometry reveals detectable intermediates in the kinetic folding pathways of each of these mutants. Comparison of the quench-flow data analyzed by NMR for the wild-type protein and the mutants showed that the substitutions I28A, L29A and L32A lead to destabilization of the B helix in the burst phase kinetic intermediate, relative to wild-type apomyoglobin. In contrast, the I30A mutation apparently has a slight stabilizing effect on the B helix in the burst phase intermediate; under weak labeling conditions, residues in the C helix region were also relatively stabilized in the mutant compared to the wild-type protein. This suggests that native-like helix B/helix C packing interactions occur in the folding intermediate. The L32A mutant showed significantly lower proton occupancies in the burst phase for several residues in the G helix, specifically F106, I107, E109 and A110, which are in close proximity to L32 in the X-ray structure of myoglobin, providing direct evidence that native-like helix B/helix G contacts are formed in the apomyoglobin burst phase intermediate. The L29A mutation resulted in an increase in burst phase proton occupancies for several residues in the E helix. Since these regions of the B and E helices are not in contact in the native myoglobin structure, these effects suggest the possibility of non-native B/E packing interactions in the kinetic intermediate. The differing effects of these B helix mutations on the apomyoglobin folding process suggests that each side-chain plays a different and important role in forming stable structure in the burst phase intermediate, and points to a role for both native-like and non-native contacts in stabilization of the folding intermediate.

Authors+Show Affiliations

Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14607120

Citation

Nishimura, Chiaki, et al. "Role of the B Helix in Early Folding Events in Apomyoglobin: Evidence From Site-directed Mutagenesis for Native-like Long Range Interactions." Journal of Molecular Biology, vol. 334, no. 2, 2003, pp. 293-307.
Nishimura C, Wright PE, Dyson HJ. Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions. J Mol Biol. 2003;334(2):293-307.
Nishimura, C., Wright, P. E., & Dyson, H. J. (2003). Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions. Journal of Molecular Biology, 334(2), 293-307.
Nishimura C, Wright PE, Dyson HJ. Role of the B Helix in Early Folding Events in Apomyoglobin: Evidence From Site-directed Mutagenesis for Native-like Long Range Interactions. J Mol Biol. 2003 Nov 21;334(2):293-307. PubMed PMID: 14607120.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions. AU - Nishimura,Chiaki, AU - Wright,Peter E, AU - Dyson,H Jane, PY - 2003/11/11/pubmed PY - 2003/12/19/medline PY - 2003/11/11/entrez SP - 293 EP - 307 JF - Journal of molecular biology JO - J Mol Biol VL - 334 IS - 2 N2 - The folding pathways of four mutants in which bulky hydrophobic residues in the B helix of apomyoglobin (ApoMb) are replaced by alanine (I28A, L29A, I30A, and L32A) have been analyzed using equilibrium and kinetic methods employing NMR, CD, fluorescence and mass spectrometry. Hydrogen exchange pulse-labeling followed by mass spectrometry reveals detectable intermediates in the kinetic folding pathways of each of these mutants. Comparison of the quench-flow data analyzed by NMR for the wild-type protein and the mutants showed that the substitutions I28A, L29A and L32A lead to destabilization of the B helix in the burst phase kinetic intermediate, relative to wild-type apomyoglobin. In contrast, the I30A mutation apparently has a slight stabilizing effect on the B helix in the burst phase intermediate; under weak labeling conditions, residues in the C helix region were also relatively stabilized in the mutant compared to the wild-type protein. This suggests that native-like helix B/helix C packing interactions occur in the folding intermediate. The L32A mutant showed significantly lower proton occupancies in the burst phase for several residues in the G helix, specifically F106, I107, E109 and A110, which are in close proximity to L32 in the X-ray structure of myoglobin, providing direct evidence that native-like helix B/helix G contacts are formed in the apomyoglobin burst phase intermediate. The L29A mutation resulted in an increase in burst phase proton occupancies for several residues in the E helix. Since these regions of the B and E helices are not in contact in the native myoglobin structure, these effects suggest the possibility of non-native B/E packing interactions in the kinetic intermediate. The differing effects of these B helix mutations on the apomyoglobin folding process suggests that each side-chain plays a different and important role in forming stable structure in the burst phase intermediate, and points to a role for both native-like and non-native contacts in stabilization of the folding intermediate. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/14607120/Role_of_the_B_helix_in_early_folding_events_in_apomyoglobin:_evidence_from_site_directed_mutagenesis_for_native_like_long_range_interactions_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022283603011938 DB - PRIME DP - Unbound Medicine ER -