Proteins are the metabolic workhorses of the
cell; they engage in a variety of essential activities ranging from
enzymatically catabolizing macromolecular food sources to serving as structural
components that maintain cell stability.  Maximizing protein function relies
on intricate non-covalent interactions occurring on the secondary, tertiary,
and quaternary levels that help determine the overall shape of the protein. In
their native states, proteins will assume the most energetically favorable
configuration.  Occasionally however, cells are exposed to exogenous disruptions
such as heat stress. Heat Stress can compromise protein three-dimensional structure.
Hydrophobic residues tend to be buried in the interior of the protein but when
exposed to stress, the previously buried hydrophobic residues can be exposed to
the hydrophilic aqueous cellular environment. This unstable conformation forces
the protein to associate with nearby polypeptides, thus forming aggregates,
which can be lethal to the cell.  In addition to denaturing proteins, newly
formed polypeptides made on the ribosome face the challenge of folding properly
within the crowded cellular environment. 

To counteract these heat stress effects, evolution
has selectively pressured organisms to have defensive capabilities for protein protection to prevent aggregation
and maintain native protein conformation. Many organisms have small heat shock
proteins (sHSP) and heat shock proteins (HSP), which are molecular chaperones,
are essential for protection. Molecular chaperone proteins are essential
components in the maintenance and turnover of proteins and they play an
integral role in the folding and unfolding of cellular protein substrates.
 Most chaperones, such as HSPs, can be characterized as either
ATP-dependent, which use the power of ATP hydrolysis to modulate domain
structure and facilitate substrate binding and release. Others may be
ATP-independent chaperones, which bind unfolding substrates, trap, and then
hold them via non-covalent interactions.  

One ATP-independent chaperone class is the Small
Heat Shock Proteins (sHSPs), which act as molecular life vests and are thought
to protect misfolding proteins from irreversible aggregation (CITATION Haslbeck & Vierling JMB 2015?).  sHSPs are a ubiquitous class of
chaperones found across all kingdoms of life. sHSP range in size from 12-42
kilo Daltons in large oligomers of 12 to >32 subunits and the structure is
homologous across all species. The sHSP monomer consists of three domains: a
disordered N-terminal arm, a beta-sandwich ?-crystallin domain, and a flexible
C-terminal extension. The N-terminal domain is the most variable region with
little conservation between species.  Experimental evidence also suggests
N-terminal involvement in  substrate
binding and protection.  The ?-crystallin domain is the most highly
conserved region and adopts a ?-sandwich conformation composed of 7 to 8
anti-parallel ?-strands (Basha et al, 2012). The C-terminus contains an I-X-I
motif, which helps to satblizie the oligomeric form of the sHSP (Basha et al,
2012).

Data from previous studies suggest that the
inactive sHSP takes on the oligomer conformation Upon stress, these oligomers assemble
into active dimeric species, exposing previously inaccessible hydrophobic
surfaces that can then interact with nonpolar patches on the misfolded
substrate, capturing them in large complexes.  The sHSP-substrate
complexes maintain the substrate in a folding-competent state for extended
periods of time. Biologically this is of utmost importance since it is
essential to maintain the partially denaturing substrates in a
folding-competent state until the stress has passed and ATP-dependent
chaperones can refold them (Jaya et al, 2009).  In addition, sHSPs may be
essential facilitators for downstream refolding, unfolding, or degradation of
some of their substrates.  However, these mechanisms are still unknown.

Synechocystis sp. PCC 6803 is an excellent model organism for the study
of sHSP function in vivo.  As a single celled photosynthetic
cyanobacterium, Synechocystis has a fully sequenced genome that can be
transformed by homologous recombination, and null loci have been identified
that can be utilized for introduction of heterologous genes.  The genome
of Synechocystis encodes only one sHSP, Hsp16.6, and it has been
shown to be essential for acquired thermotolerance.  Hsp16.6 is found in
position 460250 to 460690 of the chromosome and is annotated as sll1514
(Nakamura et al, 1998). It is 146 amino acids in length and has the same
conserved architecture as other sHSPs. Mass spectrometry data suggest that this
protein adopts a multiple higher order oligomeric species ranging from a 12mer
to a 28mer, with the predominant species being a 18mer (J. Benesch, Oxford,
U.K., personal communication).  Despite all of the descriptive data on
Hsp16.6, substrates of the sHSP have not been fully characterized.  As a
result, the goal of this work is to elucidate substrates of Hsp16.6 and in
doing so, better understand the critical roles that sHSPs play in cell
viability during stress. To accomplish this, a Synechocystis strain will
be engineered that will make it possible to crosslink Hsp16.6 to substrates in
vivo, which can then be identified by mass spectrometry.  

Benzoyl-L-phenylalanine (BPA) is a phenylalanine
analog that following irradiation with UV light (280 nm) when introduced into
Hsp16.6 forms zero-length cross-links (0 Å) with proteins in close proximity
protein substrates following irradiation with UV light (280 nm).
 Introducing BPA into Hsp16.6 will allows for covalent attachment of the
sHSP with andits target substrate or other interacting protein. This, which
will allow for purification and subsequent characterization of unknown protein
substrates.  Since BPA is an unnatural, amino acid a mutated tRNA/tRNA
synthetase combination must be employed in order to introduce BPA at the
desired residues; the codon that will specify incorporation of BPA will be the
TAG amber codon (Schultz, et al 2002).    

Objective:

The overall goal of this
project is to obtain new insight into substrate interactions of sHSPs in vivo
by studying the single sHSP, Hsp16.6, of the cyanobacterium Synechocystis
sp. PCC 6803. To accomplish this, a method previously used in Escherichia
coli (E. coli) will be adapted to Synechocystis. This will
involve two major steps: 1) introducing residue sites for BPA
incorporationcrosslinking into Hsp16.6, and 2) genetically engineering Synechocystis
to express a modified tRNA and tRNA synthetase combination that will
incorporate the cross-linking, amino acid analog BPA into the desired sites.
 

 

For the first step, tThe
immediate aims focus on using site-directed mutagenesis of the wild type
Hsp16.6 gene (with an added C-terminal Strep II affinity tag REF) in order to
insert BPA at specific positions in the sHSP.  The residues that were
chosen are the following: L5, F15, F23, S101, and K137, of which two constructs
(F15, S101, and K137) have already been successfully created and sequenced.
 These residues were chosen based on previous published work done by
colleague Nomalie Jayafrom the lab . Her These cross-linking studies were
performed with Hsp18.1, a higher plant sHSP with significant homology to
Hsp16.6, allowed us to choose residues that would most likely result in
successful cross-linking with Hsp16.6.  Another criterion that was
considered was the similarity in the chemical properties of the residues to the
BPA cross-linker. We wanted to ensure that the incorporation of a bulky
hydrophobic compound like BPA would not compromise the three-dimensional
stability of the sHSP.  Currently, co-transformations are being conducted
with the BPA mutant constructs and the pEVOL plasmid  (encoding the
tRNA/tRNA synthetase required for BPA incorporation) into competent BL21 (DE3) E.
coli cells in order to test whether or not BPA incorporation compromises
the oligomeric properties of Hsp16.6 in vitro.  

 

For Step 2, theAnother
immediate aim involves constructing a Synechocystis strain in which the
genes for the modified tRNA/tRNA synthetase combination are regulated under a
native Synechocystis promoter.  Currently the genes reside on
pEVOL, a plasmid derived from E. coli that utilizes the arabinose
promoter, which is a non-functional promoter in Synechocystis.  As
such, the goal will is be to remove the gene of interest from pEVOL and
incorporate the gene downstream of a native Synechocystis promoter into
a different vector using recombinant DNA cloning procedures.  This goal
can be accomplished through a series of restriction enzyme digestions that will
be described in more detail (see Mmethodology section).  

 

Methodology

*Note: all
measurements are given in final concentrations except those where the initial
concentration is unknown but standard.  For example, “6 uL of standard Gel
Red.”  Additionally, it is also implied that sterile technique is of
utmost importance at each step.

 

The research will be
carried out in on the third floor of the Life Science Laboratories (LSL), a
state of the art facility with the appropriate technology in order to complete
my project.  Additionally, there is a wealth of support available
to me in the form of graduate students, post-docs (from my lab and other labs),
nearby biochemistry and protein chemistry professors and of course the PI
Professor Elizabeth Vierling.  The following protocol describes