Changes in the Apomyoglobin Folding Pathway Caused by Mutation of the Distal Histidine Residue

Garcı́a, Carlos; Nishimura, Chiaki; Cavagnero, Silvia; Dyson, H. Jane; Wright, Peter E.

doi:10.1021/bi0010266

Cited by 67 publications

(88 citation statements)

References 32 publications

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“…The active-site residues of enzymes are generally polar or charged, and are usually located in hydrophobic clefts [Fersht, 1999]. Stabilizing mutations in active site residues can reduce enzymatic activities [Beadle and Shoichet, 2002;Counago et al, 2008;Garcia et al, 2000;Kidokoro et al, 1995;Meiering et al, 1992;Mukaiyama et al, 2006;Nagatani et al, 2007;Schreiber et al, 1994, Shoichet et al, 1995Zhi et al, 1991]. Additionally, a stabilizing mutation increased the resistance of ribonuclease A to proteolysis [Markert et al, 2001], which, for example, would be an undesirable effect if it occurred in enzymes involved in cell signaling [Fink, 2005].…”

Section: Discussionmentioning

confidence: 99%

Performance of protein stability predictors

2010

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Stability is a fundamental property affecting function, activity, and regulation of biomolecules. Stability changes are often found for mutated proteins involved in diseases. Stability predictors computationally predict protein-stability changes caused by mutations. We performed a systematic analysis of 11 online stability predictors' performances. These predictors are CUPSAT, Dmutant, FoldX, I-Mutant2.0, two versions of IMutant3.0 (sequence and structure versions), MultiMutate, MUpro, SCide, Scpred, and SRide. As input, 1,784 single mutations found in 80 proteins were used, and these mutations did not include those used for training. The programs' performances were also assessed according to where the mutations were found in the proteins, that is, in secondary structures and on the surface or in the core of a protein, and according to protein structure type. The extents to which the mutations altered the occupied volumes at the residue sites and the charge interactions were also characterized. The predictions of all programs were in line with the experimental data. I-Mutant3.0 (utilizing structural information), Dmutant, and FoldX were the most reliable predictors. The stability-center predictors performed with similar accuracy. However, at best, the predictions were only moderately accurate ($60%) and significantly better tools would be needed for routine analysis of mutation effects. Hum Mutat 31:675-684,

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Section: Discussionmentioning

confidence: 99%

Performance of protein stability predictors

2010

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“…Yet an experiment in which the H helix of myoglobin was mutated to decrease its helical propensity (30) showed very little influence on the folding rate of the protein. A much greater effect was observed when the distal histidine (H64) was changed to phenylalanine: the substitution of a nonpolar side chain for the buried polar histidine resulted in a significant increase in the rate of folding (31). These studies pointed toward the importance of side-chain packing, particularly in the contact surfaces of helices, for the rate of folding of apomyoglobin.…”

Section: Experimental Verification Of Modelsmentioning

confidence: 99%

The role of hydrophobic interactions in initiation and propagation of protein folding

Dyson

Wright

Scheraga

2006

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

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Globular proteins fold by minimizing the nonpolar surface that is exposed to water, while simultaneously providing hydrogenbonding interactions for buried backbone groups, usually in the form of secondary structures such as ␣-helices, ␤-sheets, and tight turns. A primary thermodynamic driving force for the formation of globular structure is thus the sequestration of nonpolar groups, but the correlation between the parts of proteins that are observed to fold first (termed folding initiation sites) and the ''hydrophobicity'' (as customarily defined) of the amino acids in these regions has been quite weak. It has previously been noted that many amino acid side chains contain considerable nonpolar sections, even if they also contain polar or charged groups. For example, a lysine side chain contains four methylenes, which may undergo hydrophobic interactions if the charged -NH 3 ؉ group is saltbridged or hydrogen-bonded. Folding initiation sites might therefore contain not only accepted ''hydrophobic'' amino acids, but also larger charged side chains. Recent experiments on the folding of mutant apomyoglobins provides corroboration for models based on the hypothesis that folding initiation sites arise from hydrophobic interactions. A near-perfect correlation was observed between the areas of the molecule that are present in the burstphase kinetic intermediate and both the free energy of formation of hydrophobic initiation sites and the parameter ''average area buried upon folding,'' which pinpoints large side chains, even those containing charged or polar portions. These results provide a putative mechanism for the control of protein-folding initiation and growth by polar͞nonpolar sequence propensity alone.folding pathways ͉ myoglobin ͉ ribonuclease F reed of the ribosome, and in the absence of chaperones, a newly synthesized protein exists in an ensemble of states, the so-called statistical coil, the nature of which is a subject of much recent investigation (1). It subsequently folds to the native biologically active structure whose free energy is considered to be a minimum under appropriate solution conditions (2). Since the seminal work of Anfinsen (2), the kinetics of the folding process and the final structure have been considered to be determined by the physical interactions among the amino acid residues to attain the thermodynamically favored state.A major question has involved the exact mechanism by which the interresidue interactions specify the initiation of folding in the unfolded polypeptide chain under folding conditions. Because nonpolar groups are not soluble in water, attention has been focused on the hydrophobic interactions between nonpolar side chains to achieve their sequestration from aqueous solvent. A model has been proposed in which nearby nonpolar groups in the chain participate in hydrophobic interactions with minimal loss of entropy of the chain because of the proximity of the interacting side chains in the amino acid sequence (3).Yet the chemical nature of the polypeptide chain complicates ...

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“…The distal histidine residue in position 64 (His E7) plays a key role in the binding of oxygen and is highly conserved among globins for functional reasons. Replacement of the rather polar histidine residue by a hydrophobic phenylalanine results in significant changes in the thermodynamic stability as well as the folding kinetics of apomyoglobin (Garcia et al 2000). The replacement of His64 with Phe is expected to have little effect on the helix propensity of the apomyoglobin E helix, according to the prediction program AGADIR (Muñoz and Serrano 1994).…”

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“…The replacement of His64 with Phe is expected to have little effect on the helix propensity of the apomyoglobin E helix, according to the prediction program AGADIR (Muñoz and Serrano 1994). Nevertheless, a significantly increased helix content is observed in the native and molten globule states by CD, fluorescence, and NMR spectroscopy, and the mutation also results in an increased thermodynamic stability (Garcia et al 2000). Stopped-flow CD experiments reveal a higher helix content in the burst phase intermediate (Garcia et al 2000), and quench-flow hydrogen exchange experiments show that helix E, which is partially populated in the conformational ensemble of the wild-type kinetic intermediate (Nishimura et al 2002), is now protected on the same timescale as helices A, B, G, and H, which form the molten globule in wild-type apomyoglobin (Garcia et al 2000).…”

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confidence: 99%

Structural characterization of partially folded intermediates of apomyoglobin H64F

et al. 2008

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We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1 H]-15 N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid-and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.

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Changes in the Apomyoglobin Folding Pathway Caused by Mutation of the Distal Histidine Residue

Cited by 67 publications

References 32 publications

Performance of protein stability predictors

Performance of protein stability predictors

The role of hydrophobic interactions in initiation and propagation of protein folding

Structural characterization of partially folded intermediates of apomyoglobin H64F

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