Proline residues in transmembrane α helices affect the folding of bacteriorhodopsin1 1Edited by A. R. Fersht

Lu, Hui; Marti, Thomas; Booth, Paula J.

doi:10.1006/jmbi.2001.4605

Cited by 62 publications

(58 citation statements)

References 30 publications

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“…We observe high ⌽ values for much of helix B (Fig. 3 and Table 1), which are consistent with previous suggestions (18,29,30) that most of this particular helix, and its structural interactions, are formed early in folding. However, the complexity of the bR refolding system gives rise to some caveats.…”

Section: Solvent Interactions and Structured Unfolded Statessupporting

confidence: 91%

The transition state for integral membrane protein folding

Curnow

Booth

2009

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

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Biology relies on the precise self-assembly of its molecular components. Generic principles of protein folding have emerged from extensive studies on small, water-soluble proteins, but it is unclear how these ideas are translated into more complex situations. In particular, the one-third of cellular proteins that reside in biological membranes will not fold like water-soluble proteins because membrane proteins need to expose, not hide, their hydrophobic surfaces. Here, we apply the powerful protein engineering method of ⌽-value analysis to investigate the folding transition state of the alpha-helical membrane protein, bacteriorhodopsin, from a partially unfolded state. Our results imply that much of helix B of the seven-transmembrane helical protein is structured in the transition state with single-point alanine mutations in helix B giving ⌽ values >0.8. However, residues Y43 and T46 give lower ⌽ values of 0.3 and 0.5, respectively, suggesting a possible reduction in native structure in this region of the helix. Destabilizing mutations also increase the activation energy of folding, which is accompanied by an apparent movement of the transition state toward the partially unfolded state. This apparent transition state movement is most likely due to destabilization of the structured, unfolded state. These results contrast with the Hammond effect seen for several water-soluble proteins in which destabilizing mutations cause the transition state to move toward, and become closer in energy to, the folded state. We thus introduce a classic folding analysis method to membrane proteins, providing critical insight into the folding transition state.phi value ͉ kinetics ͉ thermodynamics ͉ protein engineering P rotein folding plays a central role in biology. Folding investigations provide key information on protein structure and dynamics, while protein misfolding can have serious disease implications (1, 2). To fold correctly, proteins must overcome an activation barrier to pass through a high-energy transition state. Understanding the nature of this folding transition state is important in resolving how a protein folds to a stable and functional native structure (3, 4).The most powerful method available to probe the structure and energetics of the folding transition state combines sitedirected mutagenesis, equilibrium thermodynamics, and kinetic data in a ⌽-value analysis. This approach has revolutionized protein folding studies by providing a quantitative description of the environment experienced by individual side chains in the folding transition state and has been applied to the folding of many small, water-soluble proteins (3,(5)(6)(7)(8). However, the ⌽-value method has yet to be applied to larger integral membrane proteins.Integral membrane proteins are a special case in protein folding because they are adapted to the lipid bilayer rather than to the cytoplasmic milieu (9). The sequences of transmembrane regions are biased in favor of hydrophobic amino acids to match the low dielectric of the membrane interior. This...

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Section: Solvent Interactions and Structured Unfolded Statessupporting

confidence: 91%

The transition state for integral membrane protein folding

Curnow

Booth

2009

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

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“…Thus, the highly conserved Pro234 functions in a dual capacity, probably essential for both proper protein expression and function. This two-pronged contribution of prolines, especially those situated in membrane segments, has been documented previously for many other important solute carriers and exchangers as well (Lin et al, 2000;Lu et al, 2001;Shelden et al, 2001;Labro et al, 2003;Slepkov et al, 2004;Joshi and Pajor, 2006). As observed by Kaback and colleagues for Lac per- mease, a paradigm of secondary active transporters, relatively few amino acids are essential for transport unless directly involved in forming linkages with substrate; instead, such carriers must be "highly flexible proteins capable of widespread conformational changes during turnover" (Kaback and Wu, 1997).…”

Section: Discussionsupporting

confidence: 72%

Conformational Flexibility of Helix VI Is Essential for Substrate Permeation of the Human Apical Sodium-Dependent Bile Acid Transporter

Hussainzada

Khandewal²,

Swaan³

2007

Mol Pharmacol

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“…It is of crucial interest in this regard to note that the presence or absence of proline changes helix-bilayer interactions, 40,41 and has been suggested to influence BR folding. 42 Since the folding and functional state of individual membrane proteins can be determined with SMFS, 43,44 the presented approach may be applied in future to determine which energetic pathway favors membrane protein malfunction, destabilization and misfolding. At present, it is difficult to ascertain which pathways lead to the native folded state and which to the nonnative or misfolded states.…”

Section: Discussionmentioning

confidence: 99%

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

Sapra

Balasubramanian

Labudde

et al. 2008

Journal of Molecular Biology

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Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others.

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Proline residues in transmembrane α helices affect the folding of bacteriorhodopsin1 1Edited by A. R. Fersht

Cited by 62 publications

References 30 publications

The transition state for integral membrane protein folding

The transition state for integral membrane protein folding

Conformational Flexibility of Helix VI Is Essential for Substrate Permeation of the Human Apical Sodium-Dependent Bile Acid Transporter

Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

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