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Hans Merzendorfer

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Heat shock proteins are a structurally diverse class of proteins whose gene expression is usually increased in response to heat or other stress. This class includes small heat shock proteins (sHsps), which serve as ATP-independent molecular chaperones. By binding to partially unfolded proteins, they prevent or reverse harmful protein aggregation, which impairs cellular function. Accordingly, sHsp malfunctions have been connected with various diseases such as cataracts and different muscular and neurodegenerative disorders. How they fulfil this important function has been studied extensively, but now a recent paper published by Mason Posner and colleagues from various US institutions provides new insight into the function and evolution of these proteins by comparing sHSPs among bony fishes with different body temperatures.

A key feature of sHsps – as well as vertebrate α-crystallins (α-Cs), closely related eye-lens proteins with chaperone-like activity – is that they bind to exposed hydrophobic regions on the surface of partially unfolded proteins to suppress protein aggregation. This process requires an increase in the hydrophobic surface on the sHsps/α-Cs to facilitate protein binding and chaperone activity. Precisely how this increase in hydrophobicity occurs is uncertain. Posner and his colleagues hypothesized that if they analyzed the amino acid sequences of different α-Cs from teleost (bony) fish adapted to different environmental temperatures, they would be able to learn about the structural requirements for chaperone activity, because it is known that the hydrophobicity of the surface of proteins varies depending on the temperature at which they function. For example, warm-adapted proteins have a smaller fraction of hydrophobic amino acids on the surface than cold-adapted proteins, and vice versa.

First, they measured the chaperone function of six different recombinant bony fish α-Cs by testing their ability to protect insulin or lactalbumin from aggregation. They noticed that α-Cs from cooler-bodied fish showed a greater chaperone activity than the α-Cs of warmer-bodied fish at the same assay temperature. This correlation was partially a result of the different thermal stabilities of the chaperones. Next, they carefully analyzed the amino acid composition and found that the α-Cs of warmer-bodied fish had fewer hydrophobic amino acids. Finally, they identified three amino acid positions in the α-Cs, where the local hydrophobicity varied significantly across the six species, potentially affecting the chaperone's stability and activity at different body temperatures.

Predicting that they could reduce the thermal stability of α-C while increasing the chaperone activity by changing the amino acids at these three locations, the team mutated the zebrafish α-C protein accordingly and measured the mutant's stability and chaperone activity. Indeed, one of the three substitutions fitted their hypothesis, as it affected both chaperone activity and protein stability in the predicted directions. However, the other two residues did not perfectly match their prediction; they either only increased activity or reduced thermal stability. Regardless of this deviation from expectation, the results suggested that all three identified amino acids are particularly important for the function of α-C.

Posner and his colleagues have convincingly demonstrated that a comparative approach is extremely useful in the analysis of the structure–function relationship of sHsps/α-Cs. By carefully analysing α-Cs from bony fish adapted to different environmental temperatures, they have not only identified the key amino acid positions that affect the protein's stability and chaperone activity, but also provided evidence for an evolutionary mechanism that has adapted chaperone activity to different environmental temperatures through the alteration of hydrophobicity at crucial locations in the protein structure.