Conformational Heterogeneity of an Equilibrium Folding Intermediate Quantified and Mapped by Scanning Mutagenesis

Yan, S. Frank; Gawlak, Grzegorz; Smith, Jonathan; Silver, Lin E.; Koide, Shohei

doi:10.1016/j.jmb.2004.02.063

Cited by 32 publications

(60 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The NHX results and mutational phi analysis showed that at least two PUFs constructed from these foldons account for sequential intermediates in the folding pathway (Yan et al 2004). …”

Section: Outer Surface Protein a (Ospa)mentioning

confidence: 99%

“…Moreover, entire Ω-loops act as concerted unfolding units (Hoang et al 2002;Krishna et al 2003b), unsurprisingly so, because they are internally packed self-contained structures (Leszczynski & Rose, 1986). β-structures tend to break up into smaller separately cooperative units (Chamberlain et al 1996;Yan et al 2002Yan et al , 2004Bédard et al 2008). …”

Section: Foldon Structurementioning

confidence: 99%

See 1 more Smart Citation

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

172

267

View full text Add to dashboard Cite

Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. The protein folding problemThe search for protein folding pathways and the principles that guide them has proven to be one of the most difficult problems in all of structural biology. Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates only live for <1 s and they cannot be isolated and studied by the usual structural methods.The protein folding problem has been attacked by the entire range of fast spectroscopic methods which are able to track folding in real time. Much valuable information has been obtained but mainly of a kinetic nature. Kinetic methods do not describe the structure of the

show abstract

“…The NHX results and mutational phi analysis showed that at least two PUFs constructed from these foldons account for sequential intermediates in the folding pathway (Yan et al 2004). …”

Section: Outer Surface Protein a (Ospa)mentioning

confidence: 99%

Section: Foldon Structurementioning

confidence: 99%

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

172

267

View full text Add to dashboard Cite

show abstract

“…16 Mutations at positions 100, 102 and 109-116, which we predicted to reduce the separation between the two unfolding transitions, were made in addition to the E160L mutation in order to obtain a better separation of the two unfolding transitions and thus greater accuracy in ΔΔG values. 16 Urea-induced unfolding reactions were monitored simultaneously with far-UV CD and Trp fluorescence, and unfolding curves were analyzed in terms of a three-state model (N -I -U) using a global fitting method as described previously.16 The m-values for the N-I and I-U transitions (m NI and m IU ) were allowed to vary, but the total m-value (m NU = m NI + m IU ) was fixed at the average value (4.4).…”

Section: Protein Preparation and Stability Measurementmentioning

confidence: 99%

“…16 Urea-induced unfolding reactions were monitored simultaneously with far-UV CD and Trp fluorescence, and unfolding curves were analyzed in terms of a three-state model (N -I -U) using a global fitting method as described previously.16 The m-values for the N-I and I-U transitions (m NI and m IU ) were allowed to vary, but the total m-value (m NU = m NI + m IU ) was fixed at the average value (4.4). 16 The use of the constant m NU value is justified because the m-value is proportional to the total change in surface burial upon folding 59 and the spectral data (see Results) indicates minimal effects of the point mutations on the conformations of the native and unfolded states of OspA, suggesting the conservation of the amount of surface burial upon folding. This method is equivalent to the commonly used one for comparing mutants of protein that undergoes a two-state unfolding in which a single, average m-value is used for obtaining ΔΔG values (e.g.…”

Section: Protein Preparation and Stability Measurementmentioning

confidence: 99%

“…We have established methods for characterizing mutation effects on thermodynamics and the folding mechanism of OspA. 16 Recently, we have developed a set of surface mutations in the globular domains that greatly facilitates crystallization, which resulted in a 1.15-Å x-ray crystal structure. 17 Taken together, OspA represents a unique model system for investigating the energetics of the formation of flat, nonglobular β-sheets.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Hydrophobic Surface Burial Is the Major Stability Determinant of a Flat, Single-layer β-Sheet

Yan¹,

Gawlak²,

Makabe³

et al. 2007

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

Formation of a flat β-sheet is a fundamental event in β-sheet-mediated protein self-assembly. To investigate contributions of various factors to the stability of flat β-sheets, we performed extensive alanine-scanning mutagenesis experiments on the single-layer β-sheet segment of Borrelia outer surface protein A (OspA). This β-sheet segment consists of β-strands with highly regular geometries that can serve as a building block for self-assembly. Our Ala-scanning approach is distinct from the conventional host-guest method in that it introduces only conservative, truncation mutations that should minimize structural perturbation. Our results showed very weak correlation with experimental β-sheet propensity scales, statistical β-sheet propensity scales, or cross-strand pairwise correlations. In contrast, our data showed strong positive correlation with the change in buried nonpolar surface area. Polar interactions including prominent Glu-Lys cross-strand pairs marginally contribute to the β-sheet stability. These results were corroborated by results from additional non-Ala mutations. Taken together, these results demonstrate the dominant contribution of nonpolar surface burial to flat β-sheet stability even at solvent-exposed positions. The OspA single-layer β-sheet achieves efficient hydrophobic surface burial without forming a hydrophobic core by a strategic placement of a variety of side chains. These findings further suggest the importance of hydrophobic interactions within a β-sheet layer in peptide self-assembly.

show abstract

Aromatic cluster mutations produce focal modulations of β‐sheet structure

et al. 2015

Self Cite

View full text Add to dashboard Cite

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model b-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer b-sheet of Borrelia outer surface protein A (OspA). This unusual b-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the b-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of b-sheets. Surprisingly, Xray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA b-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating b-sheet conformation in protein design.

show abstract

Conformational Heterogeneity of an Equilibrium Folding Intermediate Quantified and Mapped by Scanning Mutagenesis

Cited by 32 publications

References 55 publications

Protein folding and misfolding: mechanism and principles

Protein folding and misfolding: mechanism and principles

Hydrophobic Surface Burial Is the Major Stability Determinant of a Flat, Single-layer β-Sheet

Aromatic cluster mutations produce focal modulations of β‐sheet structure

Contact Info

Product

Resources

About