Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability

Kelch, Brian A.; Agard, David A.

doi:10.1016/j.jmb.2007.04.078

Cited by 32 publications

(38 citation statements)

References 56 publications

(67 reference statements)

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“…If the increased strain in the TS occurs entirely through deformation of the Phe, then we estimate the distortion of Phe228 (modeled as a Hookean spring) in the TS must be between 8-10°. This extends previous insights into the structure of the TS, which had only implicated regions around the domain interface as being important (6,8). More importantly, the enhanced contribution of side-chain distortion to the TS provides a critical rationale for incorporating strain: the increased energetic differential between TS and N directly results in an increased kinetic stability (Fig.…”

supporting

confidence: 78%

“…Structural alignments were made using Combinatorial Extension (41). The distortion of Phe228 in TFPA has been described previously (6). The 1.85 Å structure of NAPase was reported previously (8), in which distortion of Tyr228 in NAPase could be detected even with planarity restraints during refinement (∼4.2°).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, αLP's functional stability is dictated solely by its extremely high unfolding barrier [t 1∕2 ∼ 1.2 y for N to the transition state (TS)] rather than the overall free energy difference between its native and unfolded states (5). In αLP and other family members, kinetic stability is a preferred mechanism for stability because the high unfolding energy barrier and the reduced native state dynamics result in enhanced longevity for these proteases in harsh environments, such as pH fluctuations and high protease concentrations (6)(7)(8)(9).…”

mentioning

confidence: 99%

“…Based on thermodynamic, kinetic, mutational, and computational studies of αLP and its homologs (6,8,(11)(12)(13), our group has proposed a TS structure wherein each β-barrel domain is relatively well folded but the interface between the two domains is not fully formed. Although these studies have provided a phenomenological model for the TS, an explanation for the source of the unique kinetic stability observed in αLP is still lacking.…”

mentioning

confidence: 99%

“…To investigate this linkage further, we determined the high-resolution structures of two αLP homologs, TFPA (6) and NAPase (8) that use a large pro region to catalyze folding and have extremely large unfolding barriers. These proteins exhibit the same core packing as αLP and thus also show significant distortion in Phe228 (Fig.…”

mentioning

confidence: 99%

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Functional modulation of a protein folding landscape via side-chain distortion

Kelch

Salimi

Agard

2012

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

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Ultrahigh-resolution (<1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6°distortion along the side-chain, estimated to cost 4 kcal∕mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.protein distortion | protein structure | X-ray crystallography | protein stability | protein lifetime T he physicochemical forces that determine macromolecular structure and function are generally thought to be well understood. The predominant forces include well-studied hydrogen bonding, van der Waals, and ionic interactions. By contrast, bonded terms are largely ignored as it is commonly held that bond lengths and angles are at or very close to canonical values in the native state. This view is so widely adopted that nearly all crystallographic and NMR structure determination methods include restraints to optimal values. However, distortion of covalent bonds in protein sidechains away from their low energy configurations has now been noted in several ultrahigh-resolution structures (1, 2) (Fig. 1A), but no functional role for the distortion has been ascribed. We examine the functional consequences of side-chain distortion in α-lytic protease (αLP), in which a 0.83 Å structure (2) revealed a surprisingly large distortion of the aromatic ring of a conserved Phe (Phe228) by almost 6°from planarity ( Fig. 1B; about 2.6 standard deviations from the mean in Fig. 1A).The αLP sub-family of serine proteases is one of the best-studied examples of a kinetically stable protein (3, 4); i.e. a protein that...

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supporting

confidence: 78%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Functional modulation of a protein folding landscape via side-chain distortion

Kelch

Salimi

Agard

2012

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

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Thermostable Proteins

Joo

Yoo

2009

Encyclopedia of Industrial Biotechnology

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Instability of proteins is one of the major limiting factors in industrial applications. Enhancing protein stability is one of the hottest issues in industrial biotechnology. Thorough understanding of important factors related to protein thermostability and structural features of thermostable proteins may allow successful design of protein thermostability without deleterious effects on functionality and activity. Moreover, critical view on merits and demerits of different protein engineering methods including rational design, directed evolution, and computational protein design will give a guideline to engineer proteins for industrial applications. Practically, combination of rational design or computational protein design and directed evolution can overcome the defects of each method to design a better suited protein.

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Increasing the Reaction Rate of Hydroxynitrile Lyase fromHevea brasiliensistoward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters

2014

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The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted faster with mandelonitrile. The best was HbHNL-L121Y, with a 4.2-fold higher kcat and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.

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Mesophile versus Thermophile: Insights Into the Structural Mechanisms of Kinetic Stability

Cited by 32 publications

References 56 publications

Functional modulation of a protein folding landscape via side-chain distortion

Functional modulation of a protein folding landscape via side-chain distortion

Thermostable Proteins

Increasing the Reaction Rate of Hydroxynitrile Lyase fromHevea brasiliensistoward Mandelonitrile by Copying Active Site Residues from an Esterase that Accepts Aromatic Esters

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