AST734

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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
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 (T30-40oC, salt concentration lt3,
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. gt 45oC
Psychrophile Temp. lt 20oC
Barophile High pressure
Xerophile Low water content
Halophile Salt content gt 3-10
Acidophile pH lt 5
Alkaliphilie pH gt 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
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
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
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
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
ThymineAdenine (TA) base pair
Polynucleotide backbone has charged phosphate
groups which are hydrophilic
GuanineCytosine (GC) base pair
Dashed lines indicate hydrogen bonds
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DNA secondary structure
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
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
Main determinant of DNA stability is the fraction
of CG base pairs in a given oligonucleotide
sequence
The primary difference between an AT and GC
base pair is that GC 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 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
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
Primary structure is determined by covalent amide
bonds between individual amino acids
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Proteins secondary structure
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
Tertiary and quaternary structure is defined
almost entirely by noncovalent interactions
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Protein model systems ??helices
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 Genetics 1997,
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
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What we will look at adaptive proteins
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
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
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-oligonuicleot
ides interactions
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Bringing it all together
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