Paleomolecular biochemistry Paleomolecular biochemistry: the scientific discipline devoted to the “resurrection” of an ancestral protein for the purpose of studying how its biophysical properties evolved, or to make inferences about the evolution of the organisms that expressed the protein. 1
Paleomolecular biochemistry: allows us to work beyond the limits of ancient DNA Ancient DNA Paleomolecular biochemistry Paleomolecular biochemistry: can’t do it without ancestral reconstruction Ancestral reconstruction: the inference of the ancestral character states of a gene or protein sequence for the most recent common ancestor of a given set of descendent sequences. 2
Paleomolecular biochemistry: can’t do it without ancestral reconstruction G - PR G - PR B- PR B- PR Ancestral protein 1 Ancestral protein 2 1. Identify a period of rapid genetic change, or episode of adaptive evolution ( d N / d S ). 2. Now determine if this episode in the molecules history is correlated with events in the geological, paleanotological or phylogenetic record 3. Resurrect proteins from points before and after the molecular episode. 4. Examine changes in phenotypes between the two proteins. Paleomolecular biochemistry: a generalized protocol 1. Obtain DNA/protein sequences 2. Resolve 3D structure of protein 3. Measure phenotype of modern proteins 4. Infer a phylogeny for the gene 5. Identify which of many genetic changes are adaptive (or rapid) 6. Map sites with adaptive changes to 3D structure 7. Construct hypotheses about the affect of adaptive changes on 3D structure and phenotype 8. Site-directed mutagenesis to reconstruct “ancient genes” 9. Experimental test of hypothesis by comparing function/phenotype of ancient genes with modern genes 3
Case 1: Resurrecting ancestral coral pigments Reef-building corals exhibit an amazing variety of colours: • Proteins related to the green florescent proteins (GFPs) are responsible for the colour diversity. • GFPs in high density protect endosymbiotic algae from excessive solar irradiation. • Function in low density, as in colour-morphs is unclear. Red/blue colour morphs of the great star coal Montastraea cavernosa Paleomolecular biochemistry of GFP-like proteins: Questions: What is the evolutionary mechanism of colour diversity? Is color diversity tuned by natural selection? Is there a relationship between colour and endosymbiotic algae? Why is there colour diversity in species with low density GFPs? Case 1: Resurrecting ancestral coral pigments Gene tree for the GFP-like family of proteins Red fluorescence requires much higher level of functional complexity than green or blue. Green and blue can evolve multiple times by loss of function at some sites. This is not too “hard to do” from an MRCA of all colours in M. cavernosa evolutionary standpoint. MRCA of two intermediate proteins MRCA of the RED proteins Ancestral proteins were resurrected for all four points in the phylogeny If intermediate proteins are green : red is convergent If intermediate proteins are red : red evolved only once 4
Case 1: Resurrecting ancestral coral pigments The MRCA of all colours is clearly green. The higher level of complexity in red evolved more than once. The colour of the intermediate proteins indicates that the process was a stepwise one, with incremental changes from green to red. This is a mode of adaptive evolution that was forecast by Darwin but had not been previously demonstrated in a molecular system! Bacteria were engineered to express the extant and ancestral GFP-like proteins. These bacteria were then cultured in a pattern that corresponded to the GFP-LIKE gene tree Case 1: Resurrecting ancestral coral pigments Unpublished data has revealed strong support for two patterns of adaptive evolution: 1. Episodes of change associated with colour shifts 2. Continuous modification of an area of the protein surface by diversifying selection. Microscopic view of endosymbiotic algae Colours are artificially set to maximize contrast between coral soft tissue and the endosymbiotic algae (zooxanthellae). The green colour indicates the soft tissue of the coral polyp, and the red dots indicate the endosymbiotic algal cells Novel Hypotheses : • A conflict exists between coral host and endosymbiotic algae. • Host “wants” to regulate the growth rate in its favour; algae “wants” to proliferate • Complex colour system evolved to achieve some versatility in control. • Surface is evolving under diversifying selection involved in binding algae-derived compounds 5
Case 2: Resurrecting an archosaur visual protein Example: archosaur rhodopsin protein (Chang and colleagues, 2002) Data from Chang et al. (2002) MBE 19:1493-1489. Case 2: Resurrecting an archosaur visual protein [Background] “The photoreceptors in the retina are of two types: rods and cones, so named because of their shapes. These cells are actually specialized neurons that detect light. Embedded in stacks of cell membranes in the distal portions of rods and cones are molecules that absorb certain wavelengths of light. These molecules are called photopigments and are composed of two parts: a large trans- membrane protein, an opsin, and a smaller chromophore, which is a metabolite of Vitamin A called 11-cis-retinal. The chromophore, which is embedded in the opsin, absorbs light; in so doing it undergoes a shape change. This shape change in turn activates the opsin, setting off a cascade of events that leads to a change in the electrical state of a rod or cone cell membrane. This change in the rod or cone cell membrane is then conducted via the rod or cone axon to other neurons in the retina, and from there to the brain.” [From: John Moran Eye Center, Univ of Utah. http://webvision.med.utah.edu] 6
Case 2: Resurrecting an archosaur visual protein Example: archosaur rhodopsin protein (Chang and colleagues, 2002) Data from Chang et al. (2002) MBE 19:1493-1489. Case 2: Resurrecting an archosaur visual protein Archosaur rhodopsins haven't existed for 240 million years: 1. Download existing rhodopsin gene sequences Big question: 2. Reconstruct ancestral archosaur sequence Does the protein function as a rhodopsin? 3. Reconstruct the actual gene in lab (site- directed mutagenesis) Does it bind 11-cis-retinol? Yes. 4. Put gene into an animal cell commonly Does it activate in response to light? Yes. cultured in lab Is it sensitive to visible light? Yes. 5. Gene instructs those cells to make the archosaur rhodopsin Does it interact with G-protein transducin? Yes. 6. Collect and purify the rhodopsin 7. Assess the properties of the archosaur The protein was a functioning rhodopsin! rhodopsin “Cool beans” ! 7
Case 2: Resurrecting an archosaur visual protein Rhodopsin triggers the first step in the biochemical cascade required for vision in animals - rhodopsin has a direct effect on vision - archosaur rhodopsin was red shifted - higher than most mammals and fish - within higher end of range for reptiles and birds - suggest archosaurs could see at night under dim light conditions (i.e., nocturnal) Data from Chang et al. (2002) MBE 19:1493-1489. Controversial implication : nocturnal, and not daylight, life histories might have been the ancestral state in amniotes (birds reptiles and mammals). 8
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