Loopless Rop:  Structure and Dynamics of an Engineered Homotetrameric Variant of the Repressor of Primer Protein

Glykos, Nicholas M.; Papanikolau, Y.; Vlassi, Metaxia; Kotsifaki, Dina; Cesareni, Giovanni; Kokkinidis, Michael

doi:10.1021/bi060833n

Cited by 20 publications

(39 citation statements)

References 53 publications

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“…In the presence of reducing agents, the shape of the melting curves is sigmoid, corresponding to a two-state transition between the folded and the unfolded state. Melting temperatures (T m ) were calculated from the maximum value of the first derivative of the melting curves, confirming that LLR is hyper-thermostable (T m = 92°C) in accordance with previous studies (15,16) and surpasses the stability of WT (T m = 58°C). Mutants GP (T m = 45°C), PG (T m = 40°C), PP (T m = 38°C), and A31P (T m = 36°C) are considerably less stable.…”

Section: Molten Globule States Are Established For Some Mutants In Thesupporting

confidence: 89%

“…On the other hand, the R g values for PP (23.0 ± 0.2 Å) and A31P (24.1 ± 0.4 Å) suggest less efficient packing. The R g value of LLR (32.9 ± 0.1 Å) reflects the bigger size and the elongated shape of this protein in agreement with the crystallographically determined structure (15).…”

Section: Reducing Agents Strongly Affect the Chromatographic Propertisupporting

confidence: 85%

“…The loop region and the hydrophobic core have thereby attracted particular attention, as these regions are linked with the remarkable ability of Rop mutants to adopt altered topologies and properties (10)(11)(12)(13)(14)(15). Striking examples of loop variants include mutant Loopless Rop (LLR), in which an uninterrupted pattern of heptad repeats is established through a five-residue deletion in the loop.…”

mentioning

confidence: 99%

“…Striking examples of loop variants include mutant Loopless Rop (LLR), in which an uninterrupted pattern of heptad repeats is established through a five-residue deletion in the loop. In this "loopless" mutant, the α-helical hairpin of the monomer is converted into a single helix (15,16). The complete LLR molecule is a tetramer that is completely reorganized relative to the dimeric wild-type (WT) Rop, thereby becoming a hyper-thermostable protein (16).…”

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confidence: 99%

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Structural plasticity of 4-α-helical bundles exemplified by the puzzle-like molecular assembly of the Rop protein

Amprazi

Kotsifaki

Providaki

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The dimeric Repressor of Primer (Rop) protein, a widely used model system for the study of coiled-coil 4-α-helical bundles, is characterized by a remarkable structural plasticity. Loop region mutations lead to a wide range of topologies, folding states, and altered physicochemical properties. A protein-folding study of Rop and several loop variants has identified specific residues and sequences that are linked to the observed structural plasticity. Apart from the native state, native-like and molten-globule states have been identified; these states are sensitive to reducing agents due to the formation of nonnative disulfide bridges. Pro residues in the loop are critical for the establishment of new topologies and molten globule states; their effects, however, can be in part compensated by Gly residues. The extreme plasticity in the assembly of 4-α-helical bundles reflects the capacity of the Rop sequence to combine a specific set of hydrophobic residues into strikingly different hydrophobic cores. These cores include highly hydrated ones that are consistent with the formation of interchain, nonnative disulfide bridges and the establishment of molten globules. Potential applications of this structural plasticity are among others in the engineering of bio-inspired materials.recurrent tertiary motifs | core packing | disulfide bonds | dimensionless Kratky plot R ecurrent motifs of tertiary structure are convenient model systems for studying protein folding and potentially also for the design of bio-inspired materials. For protein design purposes, structural plasticity is an important, although poorly understood, parameter to be considered, as it is among the main reasons that the re-engineering of proteins toward novel materials is not yet satisfactorily manageable (1, 2).The present study focuses on the structural plasticity associated with the 4-α-helical bundle (4HB) motif. 4HBs consist of four amphipathic α-helices packed in a parallel or antiparallel fashion (3, 4). Their folding is largely determined by a repeating pattern of hydrophobic and hydrophilic residues, organized on the basis of seven-residue repeats (heptads) (5). Being the simplest tertiary motif, 4HBs have been subject to numerous proteinfolding studies; attempts have been made to exploit them as building blocks for bio-inspired materials (6).A paradigm of a highly regular 4HB is the RNA-binding ColE1 Repressor of Primer (Rop) protein (7-9), also referred to as RNA-one-modulator (ROM). Each monomer is an α-helical hairpin consisting of two antiparallel α-helices connected by a short loop. The sequence of Rop displays a heptad repeats pattern that is interrupted only in the loop region.Structural simplicity makes Rop an attractive model system for the study of the folding of 4HBs. The loop region and the hydrophobic core have thereby attracted particular attention, as these regions are linked with the remarkable ability of Rop mutants to adopt altered topologies and properties (10-15). Striking examples of loop variants include mutant Loopless Rop...

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Section: Molten Globule States Are Established For Some Mutants In Thesupporting

confidence: 89%

Section: Reducing Agents Strongly Affect the Chromatographic Propertisupporting

confidence: 85%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural plasticity of 4-α-helical bundles exemplified by the puzzle-like molecular assembly of the Rop protein

Amprazi

Kotsifaki

Providaki

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Most of the mutants were assumed to stay in the AP arrangement because they have comparable helical content to the WT (17) and maintain their ability to bind RNA in vitro, even though some others lose it in vivo (20). X-ray and NMR measurements detected, however, three additional structures, a parallel (P, mutant Ala 2 Ile 2 -6) ¶ arrangement of helices (21), a bisecting U (BU) arrangement for a mutation in the turn region (Ala 31 Pro; data not shown) (22), and a tetrameric four-helix bundle resulting from a five-residue deletion in the turn region (23). In the cases of P and BU, the tertiary structure of the monomers maintains the WT helix-turn-helix motif.…”

mentioning

confidence: 92%

Mutations as trapdoors to two competing native conformations of the Rop-dimer

Schug

Whitford

Levy

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Conformational transitions play a central role in regulating protein function. Structure-based models with multiple basins have been used to understand the mechanisms governing these transitions. A model able to accommodate multiple folding basins is proposed to explore the mutational effects in the folding of the Rop-dimer (Rop). In experiments, Rop mutants show unusually strong increases in folding rates with marginal effects on stability. We investigate the possibility of two competing conformations representing a parallel (P) and the wild-type antiparallel (AP) arrangement of the monomers as possible native conformations. We observe occupation of both distinct states and characterize the transition pathways. An interesting observation from the simulations is that, for equivalent energetic bias, the transition to the P basin (non-wild-type basin) shows a lower free-energy barrier. Thus, the rapid kinetics observed in experiments appear to be the result of two competing states with different kinetic behavior, triggered upon mutation by the opening of a trapdoor arising from Rop's symmetric structure. The general concept of having competing conformations for the native state goes beyond explaining Rop's mutational behaviors and can be applied to other systems. A switch between competing native structures might be triggered by external factors to allow, for example, allosteric control or signaling.energy landscape ͉ protein folding ͉ conformational transition ͉ principle of minimal frustration ͉ protein function T he concept of a funneled energy landscape explains how proteins fold efficiently into a unique native conformation (1-5). A protein can fold by multiple routes in a diffusive process. Within a long evolutionary period, the shape of the energy landscape has been sufficiently smoothened to permit protein function despite environmental changes or mutations. In a funneled landscape, native interactions dominate the folding funnel, making the bias sufficiently large toward the native conformation compared with the roughness that arises from local minima. Going beyond folding, recent work has included multiple folding basins to describe conformational transitions of proteins that regulate molecular processes in biological systems or can explain, for example, the aggregation of prions (6). Approaches include thermodynamic weighting of different potentials (7), coupling of potentials (8), switching of the Hamiltonian (9), and reconstruction of contact maps (10).This article is focused on the development and application of a dual-funneled energy landscape to understand a protein's mutational behavior.The protein under investigation, the Rop-dimer (repressor of primer, Rop § ), regulates ColE1 plasmid replication in Escherichia coli through RNA binding (11)(12)(13)(14). This homodimer shows abnormal mutational behavior, as discussed below. For an interpretation within the framework of a minimally frustrated energy landscape, one must consider the symmetry of the dimer. Each monomer consists of a helix-turn-helix...

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