The Influence of Proline Isomerization and Off-Pathway Intermediates on the Folding Mechanism of Eukaryotic UMP/CMP Kinase

Lorenz, Thorsten; Reinstein, Jochen

doi:10.1016/j.jmb.2008.06.001

Cited by 14 publications

(16 citation statements)

References 59 publications

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“…Especially the increase in λ F1(RS) with urea concentration is unusual for refolding reactions. Similar observations have been made for UMPK with increases in λ 1 and λ 2 [29]. Both cases can be related to theoretical considerations by Wildegger and Kiefhaber on folding of lysozyme [30] who explain such behavior by the presence of a fast folding off-pathway intermediate that has to be unfolded before the next folding transition.…”

Section: Resultssupporting

confidence: 80%

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

2013

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The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology.

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Section: Resultssupporting

confidence: 80%

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

2013

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“…Approximately 90% of folding molecules fold via off-pathway intermediate I off , which is a relatively stable species that needs to unfold to produce native protein and thus acts as a trap. Formation of an off-pathway folding species is probably inherent to the folding of proteins with a flavodoxin-like fold [27], as this species is experimentally observed for other α-β parallel proteins of which the kinetic folding has been investigated (i.e., apoflavodoxin from Anabaena [11], CheY [28], cutinase [29], and UMP/CMP kinase [30]). …”

Section: Introductionmentioning

confidence: 92%

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

et al. 2012

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Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.

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“…[46][47][48][49] On the other hand, some proteins such as uridine monophosphate/cytidine monophosphate kinase having non-native proline residue form an off-pathway intermediate. 50 The folding of barnase is categorizable as the latter example, and the expanded conformations of barnase with the cis form of proline can be regarded as the non-productive state. We suggest that the presence or the absence of the collapse event determines the on-pathway or off-pathway mechanisms of the unfolded proteins with the non-native proline residues.…”

Section: Cis-trans Isomerization In the Folding Of Barnasementioning

confidence: 99%

Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase

Konuma

Kimura

Matsumoto

et al. 2011

Journal of Molecular Biology

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The Influence of Proline Isomerization and Off-Pathway Intermediates on the Folding Mechanism of Eukaryotic UMP/CMP Kinase

Cited by 14 publications

References 59 publications

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

Time-Resolved Small-Angle X-ray Scattering Study of the Folding Dynamics of Barnase

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