PhD Student, Australian National University
Ancestral Protein Reconstruction highlights structural changes required for emergence of catalysis
Enzymes are remarkable protein catalysts - speeding up important biochemical reactions in cells that are required for life. As protein engineers, we are always looking for ways to harness the incredible power of enzymes. For years, we have been stealing naturally occurring enzymes from living cells and using them to speed up chemical reactions in industry to make useful products - from paper to life-saving drugs. However, we are limited by the types of enzymes available in nature - it would be great if we could design and engineer our own! Advances in protein engineering mean that we can now reproduce nature's enzymes well, but whenever we try and make our own they never work very well. This highlights a gap in our understanding of how enzymes work. My research hopes to fill this gap, by studying how enzymes evolve in nature from proteins that were initially specialised for binding onto chemicals, but not for speeding up chemical reactions. We use a technique called ancestral protein reconstruction, which allows us to infer the likely amino-acid sequences of ancient ancestral forms of present-day enzymes. We study these in the lab, using X-ray crystallography to understand their atomic structures, and test for specific activities such as binding and enzyme activity. Doing this we can connect how small structural changes have allowed for the emergence and evolution of new enzymatic activity. We hope this research will help develop improved approaches to enzyme design.
Abstract: The rational design of novel biomolecules with desired functional properties is one of the most fascinating challenges in science, with implications at the fundamental and practical levels. From the fundamental point of view, the design of proteins able to support nonnatural reactivities represents the decisive test on our understanding of the molecular mechanisms through which biomolecules operate. From the practical point of view, new designs may open the way to applications in a wide variety of fields, ranging from health to life science, and from catalysis to material sciences. During the past decades, we have witnessed an amazing transition in the application of computational methods to protein and enzyme design, from simple fold prediction to ab initio structural design. Herein, we review key areas and fundamental aspects of research in the design of protein structures, interactions, and reactivities. We also provide our perspective on the exciting range of developments that are made possible by the integration of innovations in hardware, software, and theory, while keeping an eye on the applications, challenges, and opportunities that can open up in many different domains of science.For further resources related to this article, please visit the WIREs website.
Pub.: 24 May '17, Pinned: 26 Aug '17
Abstract: Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.
Pub.: 16 Mar '17, Pinned: 26 Aug '17
Abstract: A central goal in biochemistry is to explain the causes of protein sequence, structure, and function. Mainstream approaches rationalize sequence and structure by how they determine function and compare related proteins to find mechanisms underlying their functional differences. Although productive, both strategies suffer from intrinsic limitations that have left important aspects of many proteins unexplained. These limits can be overcome by reconstructing ancient proteins, experimentally characterizing their properties, and retracing their evolution through time. This approach has proven to be a powerful means for discovering how historical changes in sequence produced the functions, structures, and other physical/chemical characteristics of modern proteins. It has also illuminated whether protein features evolved because of functional optimization, historical constraint, or blind chance. Here we review recent studies employing ancestral protein reconstruction and show how they have produced new knowledge not only of molecular evolutionary processes but also of the underlying determinants of modern proteins' physical, chemical, and biological properties. Expected final online publication date for the Annual Review of Biophysics Volume 46 is May 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Pub.: 17 Mar '17, Pinned: 26 Aug '17
Abstract: Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.
Pub.: 29 Mar '17, Pinned: 26 Aug '17
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