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Fast and forceful refolding of stretched alpha-helical solenoid proteins.

ABSTRACT: Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, beta-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed alpha-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10-15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs.

SUBMITTER: Kim M

PROVIDER: S-EPMC2884255 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Fast and forceful refolding of stretched alpha-helical solenoid proteins.

Kim Minkyu M Abdi Khadar K Lee Gwangrog G Rabbi Mahir M Lee Whasil W Yang Ming M Schofield Christopher J CJ Bennett Vann V Marszalek Piotr E PE

Biophysical journal 20100601 12

Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report ...[more]

PMID: 20550922

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Project description:Membrane proteins, particularly those that are α-helical, such as transporters and G-protein-coupled receptors (GPCRs), have significant biological relevance. However, their expression and purification pose difficulties because of their poor water solubilities, which impedes progress in this field. The QTY method, a code-based protein-engineering approach, was recently developed to produce soluble transmembrane proteins. Here, we describe a comprehensive Web server built for QTY design and its relevance for in silico analyses. Typically, the simple design model is expected to require only 2 to 4 min of computer time, and the library design model requires 2 to 5 h, depending on the target protein size and the number of transmembrane helices. Detailed protocols for using the server with both the simple design and library design modules are provided. Methods for experiments following the QTY design are also included to facilitate the implementation of this approach. The design pipeline was further evaluated using microbial transmembrane proteins and structural alignment between the designed proteins and their origins by employing AlphaFold2. The results reveal that mutants generated by the developed pipeline were highly identical to their origins in terms of three-dimensional (3D) structures. In summary, the utilization of our Web server and associated protocols will enable QTY-based protein engineering to be implemented in a convenient, fast, accurate, and rational manner. The Protein Solubilizing Server (PSS) is publicly available at http://pss.sjtu.edu.cn. IMPORTANCE Water-soluble expression and purification are of considerable importance for protein identification and characterization. However, there has been a lack of an effective method for water-soluble expression of membrane proteins, which has severely hampered their studies. Here, an enabling comprehensive Web server, PSS, was developed for designing water-soluble mutants of α-helical membrane proteins, based on QTY design, a code-based protein-engineering approach. With microbial transmembrane proteins and GPCRs as examples, we systematically evaluated the server and demonstrated its successful performance. PSS is readily available for worldwide users as a Web-based tool, rendering QTY-based protein engineering convenient, efficient, accurate, and rational.

Dataset Information

Fast and forceful refolding of stretched alpha-helical solenoid proteins.

Publications

Fast and forceful refolding of stretched alpha-helical solenoid proteins.

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