Some people may not be unfamiliar with origami, in which paper is intricately folded into various shapes. But did people know that proteins in the human body also undergo a complex folding process that is critical to their structure and function?
In a new study, researchers from Northwestern University in the United States and the Institute of Industrial Science at the University of Tokyo in Japan have shed new light on protein folding stability - the tendency of proteins to maintain their folded shapes - in which protein folding stability is a central factor in diseases such as cancer, Alzheimer's disease and cystic fibrosis. They developed a new high-throughput method, cDNA display proteolysis, to assess the folding stability of nearly a million proteins in a single experiment. The results of the study were published online on July 19, 2023, in the journal Nature under the title "Mega-scale experimental analysis of protein folding stability in biology and design ".
Proteins are initially created from a single chain of amino acids, which then fold into a three-dimensional shape. Failure to properly fold or maintain this three-dimensional structure can disrupt protein function and lead to disease.
Therefore, understanding how protein folding stability is maintained will provide new ideas for studying diseases involving misfolded proteins. However, it has previously been difficult to assess protein folding stability in an efficient and large-scale manner. Therefore, these authors sought to develop a platform to assess protein folding stability in a reproducible, high-throughput manner.
Kotaro Tsuboyama, first author of the paper, said, "We started with a technique that attaches proteins to their own DNA. We generated a large number of these protein-DNA complexes using a DNA library and treated them with enzymes that destroy unfolded proteins. Intact proteins that were able to maintain their folded structure during the enzyme treatment were subsequently identified by DNA sequencing."
Image from Nature, 2023, doi:10.1038/s41586-023-06328-6
This approach allowed these authors to assess the stability of up to 900,000 protein sequences in a single test tube. To investigate how individual sequence elements in protein sequences affect folding stability, they used this approach to analyze a range of natural and artificially designed protein structural domains.
Gabriel J. Rocklin of Northwestern University, corresponding author of the paper, said, "We were able to identify a number of factors that contribute to protein stability. We also used our approach to analyze the effects of specific mutations in protein sequences and to identify determinants of stability in artificially designed proteins, thus providing new insights that will help advance future protein design methods."
Whereas previous methods for assessing protein stability have been limited to evaluating individual protein sequences, this cDNA demonstration of protein hydrolysis allows many proteins to be evaluated in a single experiment, providing an unprecedented amount of information about protein stability. This approach could advance the development of new predictive models of protein folding, thus furthering our understanding of diseases involving protein misfolding.
