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Replacement of the native cis prolines by alanine leads to simplification of the complex folding mechanism of a small globular protein

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dc.contributor.author KAUSHIK, ANUSHKA en_US
dc.contributor.author UDGAONKAR, JAYANT B.  en_US
dc.date.accessioned 2023-11-10T05:47:20Z
dc.date.available 2023-11-10T05:47:20Z
dc.date.issued 2023-10 en_US
dc.identifier.citation Biophysical Journal, 122(19), 3894-3908. en_US
dc.identifier.issn 0006-3495 en_US
dc.identifier.issn 1542-0086 en_US
dc.identifier.uri https://doi.org/10.1016/j.bpj.2023.08.012 en_US
dc.identifier.uri http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/8274
dc.description.abstract The folding mechanism of MNEI, a single-chain variant of naturally occurring double-chain monellin, is complex, with multiple parallel refolding channels. To determine whether its folding energy landscape could be simplified, the two native cis-prolines, Pro41 and Pro93, were mutated, singly and together, to Ala. The stability of P93A was the same as that of the wild-type protein, pWT; however, P41A and P41AP93A were destabilized by ∼0.9 kcal mol−1. The effects of the mutations on the very fast, fast, slow, and very slow phases of folding were studied. They showed that heterogeneity in the unfolded state arises due to cis to trans isomerization of the Gly92-Pro93 peptide bond. The Pro41 to Ala mutation abolished the very slow phase of folding, whereas surprisingly, the Pro93 to Ala mutation abolished the very fast phase of folding. Double-jump, interrupted folding experiments indicated that two sequential trans to cis proline isomerization steps, of the Gly92-Pro93 peptide bond followed by the Arg40-Pro41 peptide bond, lead to the formation of the native state. They also revealed the accumulation of a late native-like intermediate, N∗, which differs from the native state in the isomeric status of the Arg40-Pro41 bond, as well as in a few tertiary contacts as monitored by near-UV CD measurements. The Pro to Ala mutations not only eliminated the cis to trans Pro isomerization reaction in the unfolded state, but also the two trans to cis Pro isomerization reactions during folding. By doing so, and by differentially affecting the relative stabilities of folding intermediates, the mutations resulted in a simplification of the folding mechanism. The two Pro to Ala mutations together accelerate folding to such an extent that the native state forms more than 1000-fold faster than in the case of pWT. en_US
dc.language.iso en en_US
dc.publisher Elsevier B.V. en_US
dc.subject Peptide-Bond Isomerization en_US
dc.subject Ultraviolet Circular-Dichroism en_US
dc.subject Ribonuclease-A en_US
dc.subject Prolyl Isomerization en_US
dc.subject Trans Isomerization en_US
dc.subject Alpha-Subunit en_US
dc.subject Trp Synthase en_US
dc.subject Collapsed Globule en_US
dc.subject Multiple Routes en_US
dc.subject Kinetic Phase en_US
dc.title Replacement of the native cis prolines by alanine leads to simplification of the complex folding mechanism of a small globular protein en_US
dc.type Article en_US
dc.contributor.department Dept. of Biology en_US
dc.identifier.sourcetitle Biophysical Journal en_US
dc.publication.originofpublisher Foreign en_US


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