Fluoroprolines as Tools for Protein Design and Engineering

Renner, Christian; Alefelder, Stefan; Bae, Jae Hyun; Budiša, Nediljko; Huber, Robert; Moröder, Luis

doi:10.1002/1521-3773(20010302)40:5<923::aid-anie923>3.0.co;2-#

Cited by 199 publications

(165 citation statements)

References 18 publications

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“…The thermodynamic and crystallographic analyses reveal how proline derivatives can be employed favorably in other protein folds. Although we have used chemical synthesis, proline derivatives can be incorporated into proteins by heterologous expression by using proline auxotrophs of Escherichia coli or by semisynthetic methods (48)(49)(50)(51)(52)(53)(54). Derivatives other than 4-fluoroproline and 4-methylprolines can be used to bias ring pucker (55)(56)(57).…”

Section: Resultsmentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

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

153

142

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Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in ðProProGlyÞ 7 collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T m values that are increased by >50°C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 Å reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2∕3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.collagen triple helix | nonnatural amino acid | preorganization | protein stability | x-ray crystallography T he three-dimensional structures of proteins are stable because of a delicate balance of forces (1). Increases in the conformational stability of a protein-desirable in many contexts (2)-can be realized by enhancing the hydrophobic effect (3, 4), introducing or shielding a hydrogen bond (5) or electrostatic interaction (6-8), or introducing a disulfide crosslink (9-11) or metal ion-binding site (12). These strategies are often frustrated by enthalpy-entropy compensation, whereby enthalpic stabilization is offset entirely by entropic destabilization, or vice versa (13). Moreover, the strategies often lack subtlety and can lead to a misshapen and hence dysfunctional protein.As elaborated by Cram (14), the principle of preorganization states that, "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes." In a typical manifestation, a conformational constraint is imposed upon a receptor or ligand during its chemical synthesis. This principle can also yield biopolymers with increased conformational stability (Fig. 1). For example, the stability of a DNA duplex correlates with the helicity of its single strands (15, 16), and a bicyclic ("locked") derivative of ribose enhances that stability (17, 18). The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21-23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24-27).We suspected that alterations to proline residues could provide a particularly incisive means to preorganize the conformation of a polypeptide chain. In classic work, Matthews and coworkers substituted proline, which is the least flexible residu...

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Section: Resultsmentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

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

153

142

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“…The value of K Z/E is greater for the 4R series than the 4S series, which is consistent with previous work. 31,14,32,16 The effect of temperature on the values of K Z/E for 1-6 was also assessed by NMR spectroscopy. The resulting van't Hoff plots are shown in Figure 2.…”

Section: Resultsmentioning

confidence: 99%

O‐acylation of hydroxyproline residues: Effect on peptide‐bond isomerization and collagen stability

et al. 2004

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In collagen, strands of the sequence XaaYaaGly form a triple‐helical structure. The Yaa residue is often (2S,4R)‐4‐hydroxyproline (Hyp). The inductive effect of the hydroxyl group of Hyp residues greatly increases collagen stability. Here, electron withdrawal by the hydroxyl group in Hyp and its 4S diastereomer (hyp) is increased by the addition of an acetyl group or trifluoroacetyl group. The crystalline structures of AcHyp[C(O)CH3]OMe and Achyp[C(O)CH3]OMe are similar to those of AcHypOMe and AcProOMe, respectively. The O‐acylation of AcHypOMe and AchypOMe increases the 13C chemical shift of its Cγ atom: AcHyp[C(O)CF3]OMe ≅ Achyp[C(O)CF3]OMe > AcHyp[C(O)CH3]OMe ≅ Achyp[C(O)CH3]OMe ≥ AcHypOMe ≅ AchypOMe. This increased inductive effect is not apparent in the thermodynamics or kinetics of amide bond isomerization. Despite apparently unfavorable steric interactions, (ProHypGly)10, which is O‐acylated with 10 acetyl groups, forms a triple helix that has intermediate stability: (ProHypGly)10 > {ProHyp[C(O)CH3]Gly}10 ≫ (ProProGly)10. Thus, the benefit to collagen stability endowed by the hydroxyl group of Hyp residues is largely retained by an acetoxyl group. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

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“…Specifically, we generated three β2m variants in which Pro32 was replaced with (2S, 4S)-4-fluoroproline (4S-fpr), (2S, 4R)-4-fluoroproline (4R-Fpr), and 4,4-difluoroproline (F 2 Pro), respectively (16) (Fig. 1C).…”

mentioning

confidence: 99%

“…but a significantly lower activation barrier for isomerization (16). Biochemical characterization of WT β2m and the three analogs provides unique insight into the mechanistic consequences of perturbing the Pro32 peptide bond on protein (un)folding and subsequent aggregation to give oligomers and amyloid fibrils.…”

mentioning

confidence: 99%

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

Torbeev

Hilvert

2013

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

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The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cistrans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical compositionincorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein-modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation.protein conformation | polymorphism | amyloidogenesis | native chemical ligation

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Fluoroprolines as Tools for Protein Design and Engineering

Cited by 199 publications

References 18 publications

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Stereoelectronic and steric effects in side chains preorganize a protein main chain

O‐acylation of hydroxyproline residues: Effect on peptide‐bond isomerization and collagen stability

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

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