Correlation between protein stability and crystal properties of designed ROP variants

Kokkinidis, Michael; Vlassi, Metaxia; Papanikolaou, Y.; Kotsifaki, Dina; Kingswell, A J; Tsernoglou, Demetrius; Hj, Hinz

doi:10.1002/prot.340160208

Cited by 13 publications

(6 citation statements)

References 10 publications

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“…The expression, purification, crystallisation and preliminary crystallographic characterisation of A31P has been reported previously [7,25]. All crystallographic calculations were performed with the CCP4 suite of programs [22] and X-PLOR [26].…”

Section: Methodsmentioning

confidence: 99%

Protein plasticity to the extreme: changing the topology of a 4-α-helical bundle with a single amino acid substitution

1999

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Section: Methodsmentioning

confidence: 99%

Protein plasticity to the extreme: changing the topology of a 4-α-helical bundle with a single amino acid substitution

1999

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“…Replacement of loop residue Ala31 by Pro (17) results in a complete reorganization of the entire protein, which is converted from the canonical left-handed, all-antiparallel form into a right-handed mixed-parallel and antiparallel 4-α-helical bundle, displaying a "bisecting U" topology that is to a large extent determined by the local conformation at residue 31 (18). Mutant A31P displays two variations of the bisecting U topology; these differ in the relative juxtaposition of the α-helices (19).…”

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confidence: 99%

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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“…The apparent structural simplicity of its folding motif led several groups to believe that Rop could be used as a model system to investigate the sequence−structure relationships in the folding and dynamics of four-α-helix bundles in general ( , ). The result is a large body of knowledge comprising functional ( − ), thermodynamic ( − ), kinetic ( − ), and structural ( − ) investigations of many Rop mutants and variants. Unfortunately, the structural simplicity of Rop proved to be deceiving; if there is a consistent lesson to be learned from these sequence−structure studies, it is our consistent failure to understand the dependency of the Rop fold on its sequence.…”

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

Glykos¹,

Papanikolau²,

Vlassi³

et al. 2006

Biochemistry

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The repressor of primer (Rop) protein has become a steady source of surprises concerning the relationship between the sequences and the structures of several of its mutants and variants. Here we add another piece to the puzzle of Rop by showing that an engineered deletion mutant of the protein (corresponding to a deletion of residues 30-34 of the wild-type protein and designed to restore the heptad periodicity at the turn region) results in a complete reorganization of the bundle which is converted from a homodimer to a homotetramer. In contrast (and as previously shown), a two-residue insertion, which also restores the heptad periodicity, is essentially identical with wild-type Rop. The new deletion mutant structure is a canonical, left-handed, all-antiparallel bundle with a completely different hydrophobic core and distinct surface properties. The structure agrees and qualitatively explains the results from functional, thermodynamic, and kinetic studies which indicated that this deletion mutant is a biologically inactive hyperstable homotetramer. Additional insight into the stability and dynamics of the mutant structure has been obtained from extensive molecular dynamics simulations in explicit water and with full treatment of electrostatics.

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Correlation between protein stability and crystal properties of designed ROP variants

Cited by 13 publications

References 10 publications

Protein plasticity to the extreme: changing the topology of a 4-α-helical bundle with a single amino acid substitution

Protein plasticity to the extreme: changing the topology of a 4-α-helical bundle with a single amino acid substitution

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

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

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