Carol Eunmi Lee University of Wisconsin - PowerPoint Presentation

Carol Eunmi LeeUniversity of WisconsinEvolution 410 Protein Evolution

Outline(1) Types of Mutations that could affect functionStructural ChangesRegulatory Changes (2) Case study of structural changes ( protein evolution)  and how it affects temperature adaptation of the enzyme LDH

Outline(1) Types of Mutations that could affect functionStructural ChangesRegulatory Changes

Previously, we had delved into some aspects of molecular evolution when we discussed Mutations Mutations : any change in the genetic code, including those that arise from errors in DNA replication or errors in DNA repair

Most Mutations have no Effect (most in eukaryotes mutations are neutral) 3.12 billion nucleotides in the human genome Most of the genome is non-coding sequence and has no function (up to 95%): Mutations here are “Neutral” Mutations that affect function are what matter (within genes, or within regulatory sequences that affect the expression of genes)

MutationsSo, where in the genome do the mutations matter???What exactly are these mutations doing?

So, let’s go into more detail on what exactly these mutations are doing…And how they affect expression and function of genes and proteins

Diagram of eukaryotic gene

So… different types of mutations could include:STRUCTURAL: Affects the Property of the gene product (changes to the allele itself) A mutation could occur within the coding sequence of a gene, and change the amino acid composition of a protein (structural) REGULATORY: Typically affects the Amount of the gene product A mutation could occur within a regulatory element, like a promoter or enhancer near the gene ( cis -regulatory) A mutation could occur within a regulatory element, like a transcription factor that is encoded far away from the gene (trans-regulatory)

STRUCTURAL Primary: Amino Acid composition (Amino Acid substitutions) Secondary, Tertiary, Quaternary structure REGULATORY Protein expression (transcription, RNA processing, translation, etc) Protein activity (allosteric control, conformational changes, receptors) Hierarchical processes that are affected by Mutations

Once these mutations have occurred, creating genetic variation, selection could then act on genes, gene expression, and on genetic architecture (allelic and gene interactions)

STRUCTURAL evolutionary changesMutations in DNA or mRNA that result in changes in the amino acid composition of a protein Some amino acid changes alter the activity and/or function of proteins (and enzymes)

“The Central Dogma” of Molecular Biology Francis Crick (1958)

Codon Bias In the case of amino acids Mutations in Position 1, 2 lead to Amino Acid change Mutations in Position 3 often don’t matter

REGULATORY CHANGESThe protein structure itself does not changeChanges in Gene ExpressionChange in amount of expression (amount of protein made - transcribed or translated) Changes in location, timing of expression

REGULATORY Protein Expression Transcription : Mutations at promoters , enhancers ( CIS), transcription factors (TRANS), etc RNA Processing : Mutations at splice sites, sites of polyadenylation , sites controlling RNA export Translation : Mutations in ribosomes , regulatory regions, etc Protein activity ( allosteric control, conformational changes, receptors)

Gene ExpressionFocusing on Transcription alone: Cis -regulation (at or near the gene) Examples: Promoters Enhancers Local repressor Trans -regulation (somewhere else in the genome) Examples: Gene regulatory proteins (trans-acting factors, like transcription factors) Reading by Emerson et al. -- Focus reading mainly on Part 1 (Intro) and Part 2

Gene ExpressionFocusing on Trans-Acting Factors: Transcription Factors: proteins that bind to specific DNA sequences, thereby controlling transcription and gene expression. Usually regulates many genes and therefore often has large pleiotropic effects

Gene Regulation (expression) (promoter or enhancer) trans -acting factor (e.g. transcription factor) gene is expressed ACAGTGA

How do you get a new gene?Review the lecture on mutations

Paralogs: duplicated genes in a genome, followed by differentiation Orthologs: homologous genes in different populations or species

A Result of Structural Evolutionary Changes Protein Evolution

A Classic Example: temperature adaptation in Fundulus heteroclitus

LDH

LDH is a glycolytic enzyme which catalyzes the reaction between Pyruvate and Lactate

Protein functionSTRUCTURE Amino acid composition (AA substitutions) Secondary, Tertiary, Quaternary structure REGULATORY Protein expression (transcription, translation, etc) Protein activity (allosteric control, conformational changes, receptors)

Fundulus heteroclitus Populations in Maine and Georgia have different proportions of alleles (isozymes) at LDH-B

In cold temperature generally activity of an enzyme generally slows downSo, in cold temperature, enzymes generally compensate, to make up for the slower function. How? In hot temperature, enzymes have higher activity, but can denature more readily. Temperature Adaptation and Enzyme Function

Example: Enzyme Functional Evolution

Enzymes are proteins that lower the activation energy (E a ) of a chemical reaction (“catalyzes the reaction”) Different isozymes (enzymes encoded by different alleles) with different properties would lower the activation energy to differing degrees That is, enzymes with different K M or k cat will lower E a to differing degrees

Example: Enzyme Functional EvolutionImportant properties of the enzyme that could evolve:KM V max k cat = V max [E]Catalytic Efficiency

Example: Enzyme Functional EvolutionImportant properties of an enzyme that could evolve: K M : an inverse measure of the substrate's affinity for the enzyme (small K M indicates high affinity) V max : maximum reaction velocity k cat = V max Concentration of Enzyme sites Catalytic Efficiency = k cat / K M

E + S E + P E  S k 1 k 2 k -1 Enzyme Reaction E = enzyme S = substrate P = product where E  S = enzyme-substrate complex k 1 , k -1 , k 2 = enzyme reaction rates k 2 is also called k cat , the catalytic constant

Michaelis-Menten Equation V elocity (rate of reaction, dP /dt) = K M = substrate affinity; the substrate concentration where V max /2 Also called “Michaelis-Menten constant” [S] = substrate concentration V max = maximum velocity V max [S] K M + [S]

Michaelis-Menten Equation V elocity (rate of reaction) = Small K M : enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. (greater substrate binding specificity) Large K M : Need high substrate concentrations to achieve maximum reaction velocity. V max [S] K M + [S]

Catalytic EfficiencyCatalytic constant , k cat : k cat = turnover number = the rate at which substrate is converted to product, normalized per active enzyme site; E t is the concentration of enzyme sites you've added to the assay High k cat  greater rate of reaction The ratio of k cat / K M is a measure of the enzyme’s catalytic efficiency V max [E] t k cat =

E + S E + P E  S k 1 k cat k -1 Enzyme Reaction E = enzyme S = substrate P = product where E  S = enzyme-substrate complex k 1 , k -1 , k 2 = enzyme reaction rates k 2 is also called k cat , the catalytic constant K M

Video with good explanation of KM https://www.youtube.com/watch?v=rCVRC-AQ54M How the Michaelis-Menten equation is derived: https://www.youtube.com/watch?v=FXWZr3mscUo

E + S E + P E  S k 1 k cat k -1 Enzyme Reaction There could be evolutionary differences in K M or k cat in different habitats And K M and k cat among populations or species could evolve k cat depends on the  G (activation free energy) of the chemical reaction K M

In cold temperature generally activity of an enzyme generally slows downSo, in cold temperature, enzymes generally compensate, to make up for the slower function. How? –increase in k cat  increase in rate of reaction but, K M will increase (lower structure integrity) In hot temperature, enzymes have higher activity, but can denature more readily. —want to increase stability (lower k cat , lower K M ) Temperature Adaptation and Enzyme Function

1° latitude change = 1°C change in mean water temperature Place and Powers, PNAS 1979

Place and Powers, PNAS 1979 Different alleles ( isozymes ) predominate in North vs South North: LDH-B b allele (cold-adapted) South: LDH-B a allele (warm-adapted) The two alleles (proteins) differ at 2 amino acids

Place and Powers, 1979 b allele homozygote a allele homozygote Catalytic efficiency ( k cat /K M ) is higher for the b allele at low temperature, and higher for the a allele at higher temperature k cat /K M

Place and Powers, 1979 The two allele products (the enzymes) show genetic differences in catalytic efficiency (adaptive differences) across temperatures They also show Genotype x Environment interactions and evolutionary tradeoffs in function across different temperatures, with the bb homozygote doing better in the cold, and the aa homozygote doing better at higher temperature Catalytic efficiency ( k cat /K M ) is higher for the b allele at low temperature, and higher for the a allele at higher temperature k cat /K M

There are many possible limitations (costs or constraints) preventing complete adaptation to an environment due to evolutionary tradeoffsFor enzyme function, there is often a tradeoff between functional capacity (indicated by V max ) and enzyme stability (K M is one measure)

Evolutionary Tradeoff in enzyme function at cold vs high temperaturesTradeoff between flexible vs stable enzyme structure Cold Temperature: Flexible, can have higher activity to compensate for cold temperature (higher k cat ) But hard to maintain structural integrity at high temperature Warm Temperature: Stable, to maintain structural integrity at high temperature But, lower enzyme activity (ok, because temperature is high)

In damsel fish LDH, a tradeoff between functional capacity and enzyme stability has been found More cold-adapted enzymes are labile (flexible, higher k cat ) and less stable at higher temperatures More warm-adapted enzymes have been found to be more stable, but less flexible

In damsel fish LDH, a tradeoff between functional capacity and enzyme stability has been found More cold-adapted enzymes are labile (flexible, higher k cat ) and less stable at higher temperatures If too unstable, lose geometry for ligand recognition and binding (higher K M ) Protein could become inactivated

Tradeoff between functional capacity and enzyme stability Dark areas experience conformational changes during ligand binding, such that amino acid changes here could affect enzyme function ( k cat or K M ) This Thr -> Ala amino acid substitution corresponds to temperate -> tropical shift A 4 LDH

This Thr -> Ala amino acid substitution, at position 219 in the  J-  1G loop of A 4 LDH, corresponds to temperate -> tropical shift in Damselfish Threonine is more hydrophilic and thought to make the loop more flexible (higher K m , k cat )

Threonine -> Alanine amino acid substitution at a catalytic loop corresponds to temperate -> tropical shift in Damselfish Johns and Somero 2004 Chromis caudilis (tropical, warmer) Chromis punctipinnis (temperate, colder) Chromis xanthochira (tropical, warmer) Higher reaction rate in colder fish Lower stability in colder fish K M k cat

Threonine -> Alanine amino acid substitution at a catalytic loop corresponds to temperate -> tropical shift in Damselfish K M and k cat are higher in the temperate (colder) ortholog Threonine is more hydrophilic and thought to make the loop more flexible (higher V max ) The Alanine amino acid substitution causes K M and k cat to be reduced in the tropical orthologs Johns and Somero 2004 Chromis caudilis (tropical, warmer) Chromis punctipinnis (temperate, colder) Chromis xanthochira (tropical, warmer) Higher reaction rate in colder fish Lower stability in colder fish K M k cat

Threonine -> Alanine amino acid substitution at a catalytic loop corresponds to temperate -> tropical shift in Damselfish K M and k cat are higher in the temperate (colder) ortholog Johns and Somero 2004 Chromis caudilis (tropical, warmer) Chromis punctipinnis (temperate, colder) Chromis xanthochira (tropical, warmer) Higher reaction rate in colder fish Lower stability in colder fish K M k cat

KM and k cat are higher in the cold-adapted ortholog Chemical reactions slow down in colder temperature environments  Need a more flexible enzyme With a high rate of reaction to compensate for the slowed down rates of reaction in the cold In cold habitats, you need to compensate for lower rates of reaction activity by making the enzyme more flexible  high k cat sacrifice substrate affinity (high K M ) or, fast &sloppy enzymes; the cold temperature will keep the floppy enzyme more stable Higher enzyme reaction rate in cold water fish Lower enzyme stability in cold water fish K M k cat

KM and k cat are lower in the warm-adapted ortholog Chemical reactions are faster in warm temperature environments Need a more rigid enzyme, so it does not denature in the heat Such an enzyme will have a lower rate of reaction Warmer (black square, triangle): Less flexible (low k cat ), but higher binding ability (low K M ) Lower enzyme reaction rate in warm water fish Higher enzyme stability (low K M ) in warm water fish K M k cat

Tradeoffs between Enzyme lability and stability: Colder (white circles): more flexible (high k cat ), but loss of binding ability (high K M ) Warmer (black squares, triangles): Less flexible (low k cat ), but higher binding ability (low K M ) Johns and Somero 2004 Chromis caudilis (tropical, warmer) Chromis punctipinnis (temperate, colder) Chromis xanthochira (tropical, warmer) Higher reaction rate in colder fish Lower stability in colder fish K m k cat

SummaryRates of enzyme reaction speed up in higher temperatures and slow down in colder temperaturesIn low temperatures need greater enzyme lability to compensate for slower reaction rates in the cold selection will favor enzymes with higher K M and higher k cat (higher Vmax)In high temperatures need greater enzyme stability (to prevent denaturation at high temperatures)  selection will favor enzymes with lower K M and lower k cat

Patterns of Molecular EvolutionWhat are mutations? How would structural vs regulatory mutations affect function?

What are the possible targets of selection for LDH in response to temperature? How does temperature affect Enzyme Kinetics? What changes in enzyme function might enhance a response to an environmental variable (such as temperature)? (V max , K M , k cat , k cat /K M ) Why are there tradeoffs between enzyme reaction rates (functional capacity) and stability? Why are there tradeoffs between cold and warm adaptation in enzyme function? How might organisms evolve in response to global warming? What about global cooling?

1. When comparing DNA sequences that encode a protein between two species, the number of substitutions at nonsynonymous was found to be much higher than those at synonymous sites. This result suggests evidence for:(a) Non-adaptive evolution(b ) Adaptive evolution ( c ) Negative selection ( d ) Evolutionary constraint(e) Preferential fixation of synonymous sites

2. Which of the following is FALSE regarding the functional differences among the enzymes above?(a) The different genotypes appear to show tradeoffs between functioning well (higher catalytic efficiency) at cold vs warmer temperatures ( b ) The performance of the three genotypes shows no evidence for heterozygote advantage ( c ) Adaptation to temperature in these enzymes is likely due to differences in amino acid composition between the proteins encoded by the a versus b alleles ( d ) k cat /K m is higher for the aa genotype than for the bb genotype at warmer environments(e ) Differences in allelic function above reflect structural evolutionary changes and prove that regulatory changes have not occurred The graph shows the catalytic efficiency ( k cat /K m ) for three genotypes of the LDH-B enzyme across temperatures for populations of the fish Fundulus heteroclitus .

3. A fragment of DNA from an LDH-B allele shows higher number of nonsynonymous relative to synonymous substitutions than expected. When this fragment is injected into a fish, it shows elevated pyruvate metabolism relative to the equivalent fragment from another allele. (a) Evolutionary Adaptation (structural change)(b ) Evolutionary Adaptation (regulatory change) ( c ) Linkage ( d ) Physical/functional constraint(e) Insufficient information to determine

4. Which enzyme would best compensate for the challenges of living in a warmer environment?(a) A rigid enzyme (b) An enzyme with a comparatively higher Vmax (c) An enzyme with a high Km (d) A more labile enzyme with a low Vmax (e) An enzyme with a lower kcat /Km

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