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Coming Soon to a Lab Near you…. Biomolecular Adaptations to Extreme Environments. AST734 Andrew Boal 25 November 2004. Image credits: NASA, NPS, and Protein Data Bank. Extreme environments and astrobiology.

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Coming Soon to a Lab Near you…

Biomolecular

Adaptations to

Extreme

Environments

AST734

Andrew Boal

25 November 2004

Image credits: NASA, NPS, and Protein Data Bank


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Extreme environments and astrobiology

Terrestrial examples include hot springs (high temp.), salt lakes (high salt), deep sea vents (high pressure), deserts (low water)

These “extreme” environments might model conditions found on Mars, Europa, Titan, elsewhere

Numerous extreme terrestrial habitats are seen as potential analogs to life-bearing niches in the solar system

Extreme environments are those which exist outside of the conditions of a “mesophilic environment” (T~30-40oC, salt concentration <3%, etc)

Image credits: NASA, NPS


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Extreme environments: microbes in residence

Extremeophiles are defined by the type of environment required for growth

There is no overall consensus on the definition of an extreme environment

Organisms that can survive in an extreme environment but do not require those conditions for growth are extremeotolerent

Mesophile: Lives in an ambient environment

Thermophile: Temp. > ~45oC

Psychrophile: Temp. < ~20oC

Barophile: High pressure

Xerophile: Low water content

Halophile: Salt content > 3-10%

Acidophile: pH < 5

Alkaliphilie: pH > 9

Radiophile: high amounts of radiation

Image credit: CDC


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Biogeography

Biogeography is the study of the environmental distribution of species

One can explore several, isolated, analagous extreme environments which may not allow transport of microbes between them to develop a better understanding of microbial evolution

Map credit: CIA World Factbook, Image credits: NPS

But, what about a deeper look?


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Molecular components of cells

Nucleotides: protein blueprints and fabrication

Proteins: do the work of the cell

The predominant components of the molecular makeup of cells include lipids, nucleotides, and proteins

Lipids: provide cell membranes

The ability of these molecules to function is directly related to molecular shape, which is influenced by the environments, so…


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Biomolecular structural endemism

The Big Questions:

Are there molecular structures which are endemic in an environment?

If so, how and why are those structures arrived at?

What are biomolecules?

Photo Credits: National Park Service Web pages


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Biomolecular structure

Biomolecular structure is determined by a combination of covalent and noncovalent bonds

Covalent bonds are static entities which are little effected by environment

Noncovalent bonds (hydrophobic interactions, hydrogen bonding, and electrostatic attraction) exist in a dynamic equilibrium, and thus can be attenuated by factors such as temperature, ion content, and pH

Biomolecules must both be somewhat flexible and somewhat rigid to attain proper functioning, therefore the forces that hold the molecular shape must attain a balance with the environment

Too static- function is compromised

Balance- function and function preserved

Too dynamic- structure is compromised


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Lipid structure

Hydrophilic head group

H2O

Hydrophobic tail

Lipids are made up of a hydrophilic (water-loving) head group and a hydrophobic (water fearing) tail

In cell membranes, lipids pack to form a bilayer so that the heads are in water and the tails are mixed together


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Lipids in thermal environments

Thermophile Archaea bilayer

Increased hydrocarbon branching- increased hydrophobicity

Mesophile lipid bilayer

Head-tail linkages are ethers, not esters, and are chemically more robust

Lipids from thermophilic archaea have a dramatically different chemical structure

Hyperthermophile Archaea lipid

Backbone of both layers is chemically connected, again increased stability


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DNA and RNA: chemistry

Nucleobases:

Sugars:

Thymine (DNA only)

Uracil (RNA only)

Cytosine

Ribose is in RiboNucleic Acid (RNA)

Deoxyribose is in DeoxyriboNucleic Acid (DNA)

Nucleobases are cyclic structures which are basic (like ammonia)

The extra -OH (alcohol) in ribose makes it much less chemically stable

Guanine

Adenine

DNA and RNA are polymers of nucleotides (oligo- or polynucleotide)

Nucleotides are comprised of nucleobases attached to a sugar


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DNA and RNA: polynucleotide structure

DNA and RNA structure is based on hydrophobic interactions and hydrogen bonding

Hydrogen bonding is a weak interaction where two electronegative elements “share” a hydrogen atom (note that carbon-hydrogen bonds do not partake in hydrogen bonding

Center of duplex is hydrophobic

Thymine:Adenine (T:A) base pair

Polynucleotide backbone has charged phosphate groups which are hydrophilic

Guanine:Cytosine (G:C) base pair

Dashed lines indicate hydrogen bonds


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DNA: secondary structure

hairpin

duplex

junction

Base pairing determines the nature of the secondary structure

The basic elements of DNA secondary structure are the duplex (which is by far the most prevalent), the junction, and the hairpin


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DNA melting

Example of a DNA melting curve obtained spectroscopicly

Figure taken from: Drukker, K., et. al. J. Phys. Chem. B. 2000, 104, 6108-6111

Representation of DNA melting by duplex unzipping or unwinding

One of the easiest ways to measure DNA stability is to obtain a “melting curve” which is a spectroscopic measurement of duplex unzipping


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Stability of DNA in extreme environments

A:T

G:C

Main determinant of DNA stability is the fraction of C:G base pairs in a given oligonucleotide sequence

The primary difference between an A:T and G:C base pair is that G:C has three hydrogen bonds, and is thus more stable

Data taken from: Owczarzy, R., et. al. Biochemistry, 2004, 43, 3537-3554.

Other factors include hydrophobicity and interaction between salt and the DNA backbone


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The many faces of RNA

RNA is primarily involved in protein synthesis and comes in three major types:

Growing protein

Ribosome RNA (rRNA) forms the skeleton of the ribosome, the machine which makes proteins

Amino acid

Transfer RNA (tRNA) transports amino acids into the ribosome

Message RNA (mRNA) is made by transcription of DNA and lists the amino acid sequence of a protein


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Structure of tRNA

tRNA molecules are usually fairly small (less than 100 nucleic acid monomers)

tRNA has a relatively simple secondary structure

tRNA usually exists as free molecule in the cell

tRNA is a good molecule to explore for environmental studies

Like DNA, RNA secondary structure has elements such as duplexes, loops, bulges, and hairpins


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Stability of tRNA

Calculated ∆Gf values of the GGC codon tRNA from E.coli. and T. acidophilum

E. coli.: ∆Gf = -28.9 kcal/mol

T. acidophilum.: ∆Gf = -30 kcal/mol

Thermoplasma acidophilum: GGGCCGGTAGATCAGAGGTAGATCGCTTCCTTGGCATGGAAGAGGcCAGGGGTTCAAATCCCCTCCGGTCCA

E. coli.: GGGGCTATAGCTCAGCTGGGAGAGCGCTTGCATGGCATGCAAGAGGtCAGCGGTTCGATCCCGCTTAGCTCCACCA

The stability of tRNA can be both measured spectroscopicly like DNA but can also be calculated

Calculated free energy is obtained by factoring in the strength of noncovalent interactions in a folded and unfolded tRNA and is expressed as the free energy of complex formation, ∆Gf (NOTE: lower ∆Gf value indicates increased stability, formation is more favorable)

Initial ∆Gf values and predicted secondary structure can be calculated from raw sequence data:


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Proteins: amino acids and primary structure

Examples of amino acids

Amino acids

Amino functionality

Acid functionality

Side chain “R-group”- defines the chemical and physical nature of the amino acid

A peptide or protein is a chain of 10-1000 amino acids

Primary structure is determined by covalent amide bonds between individual amino acids


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Proteins: secondary structure

The -helix is a coil of a peptide chain and has 3.6 residues per helical turn

Primary interactions are hydrogen bonding between residues along the helical axis and steric interactions between side chains

Secondary structures (folds) are defined by hydrogen bonding and steric interactions of the side chain

The -sheet is a linear arrangement of amino acids

Structure is defined by inter-strand hydrogen bonds, less by sterics of side chains

Sheets can be parallel or anti-parallel, defined by orientation of the backbone

Other, but far less common, peptide folds include the coiled-coil, random coil,  bulge,  -turn, 310 helix, 27 helix, -helix, -barrel, and so on…


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Proteins: tertiary and quaternary structure

Quaternary Structure: the assembly of multiple protein units into a larger structure

Tertiary Structure: the overall shape of a folded protein

Top View

Side View

Tertiary and quaternary structure is defined almost entirely by noncovalent interactions


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Protein model systems: helices

Structural stability is measured by spectroscopicly observing helix unfolding

The -helix is a common protein structural element which can be readily studied

helices are the secondary structural element which is most susceptible to sequence and environment factors and the stability of helices is related to the stability of the overall protein

Like DNA melting, helix (and protein) stability is related to a structural denaturation

Graph taken from: Whitington, S. J., et. al. Biochemistry, 2003, 42, 14690-14695.

As for tRNA, ∆Gf can be calculated for helices or can be measured using Circular Dichrosim spectroscopy by employing the relationship ∆Gf = -RTlnK, where K can be measured from the spectrum


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Example of environment related structural differences

One example is the study of the helices of RecA

RecA is a protein involved with DNA repair, cell division and other processes and is found in all environments

Crystal structure of RecA from E. coli was used as a template

Crystal structure of RecA from E. coli

RecA sequences from 29 proteins were aligned with that of E. coli, allowing for the determination of helical fragments

There are 10 helical regions in RecA

∆Gf values for these sequences were calculated and analyzed

This work was published as: Petukov, M. et. al. Proteins: Structure, Function, and Genetics1997, 29, 309-320


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Thermophile helices are more stable

Calculated ∆Gf values indicated that helices of thermophlie origin were more stable than mesophile helices

Eight of the thermophile helices were found to be more stable- these helices are likely related to STRUCTURAL stability

No change was found for two helices, both of which are directly involved in interactions with DNA and other proteins, these helices likely need to retain flexibility for FUNCTIONAL stability

T. thermophilus (80oC)

Total helix ∆Gf

E. coli (37oC)

P. areuglinosa (20oC)

20oC

37oC

80oC

Interestingly, total helix stability was found to be the same value if the optimal temperature for protein activity is taken into account- this is again related to the need for molecular flexibility


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Biomolecular structural endemism

The Big Questions:

Are there molecular structures which are endemic in an environment?

If so, how and why are those structures arrived at?

Photo Credits: National Park Service Web pages


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Study roadmap

Bioinformatics

Sample Collection

Develop comprehensive listing of known protein/RNA sequences from public database

Identify environments for study (Hawaii lakes, Chile: Andes and Patagonia?)

Travel/Sample Collection/ Data Analysis

Search for environment-specific structural elements

Model Studies

Synthesis of short RNA and peptide sequences

Study structure of these molecules in lab-generated extreme (thermal/salt/pressure) environments

Computer models of these systems


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Environments to be explored

Initial work will be carried out in Hawaiian lakes

These include Lake Kauhako (Moloka’I), Lake Wai’ele’ele (Maui), Green Lake and lake Waiau (Hawai’I)

These lakes are relatively accessible and will provide a ready data set that we will use to develop our sampling and analysis methodologies

This data set will also establish part of the mesophile baseline

South American Lake Environments

South America, specifically the Andes and Patagonia, have numerous extremeophilic environments

South American lakes are less well studied from the biogeographical view point- will be able to describe new environments

These environments are also geographically isolated from other extreme environments will allow for greater geographic variability

Other possible environments include deep sea trenches and subglacial lakes- UH collaborations


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What we will look at: “adaptive” proteins

Antifreeze protein: inhibits ice crystal formation

Mechanosensitive channel: responds to osmotic stress

ATPsulfurylase: critical in sulfate reducing bacteria

Proteins which serve a function adapted to the environment

Potassium Channel: transports K+ into the cell


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What we will look at: “conserved” proteins

ATPase: synthesis of ATP, a cell energy source

DNA gyrase: involved in DNA packaging

Pyruvate kinase: involved in glycolysis

Rhodopsin: light sensing and transduction

Conserved proteins are those which would be expected to be more similar given a function which is ubiquitous


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Planned Methodologies: Bioinformatics and Sample Collection

Bioinformatics is the term used to describe the mining of biological sequence and structural data bases

The initial work here will be to develop a database of molecular sequences correlated with the organism of origin (which will tell us the nature of the environments they came from)

These sequences will then be examined for environment-specific structural motifs

This database will help to establish environmental targets and can be modified by biogeographical studies

Data that will be collected in the environment

Environmental DNA- will be used to establish the biodiversity of a site as well as provide information regarding molecular sequences

Physical factors will also be taken into account, including the temperature, salinity, nutrient composition, etc…


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Methodologies: Model systems and computations

tRNA sequences

helices

Synthesis and physical or computational characterization of model and natural peptides or nucleotide sequences

More complicated peptides such as the helix bundle (common in membrane proteins)

These studies will provide us with a numerical quantity (∆Gf) for stability as well as molecular level insights of the mechanism of stability

Other variants of this work includes the study of the folding of proteins isolated from the environment and the study of peptide-oligonuicleotides interactions


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Bringing it all together

This range will indicate stability window

Increasing structural similarity

Increasing stability

or protein activity

We will attempt to establish a relationship between the physical environment, biodiversity, and molecular structure

One way this can be accomplished is to generate plots of stability vs. structural similarity for individual environments

A small stability range would indicate that there are rigorous energetic requirements

This range will indicate the variance of structures which are capable of surviving

A small structural similarity range would indicate environment specific structures

If both values are small, it may indicate that structures evolved to meet the specific requirements of that environment


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