Effects of a Glycine Substitution in the Folding Nucleus of the Amyloid Protein

1
Effects of a ß-Glycine Substitution in the
Folding Nucleus of the Amyloid ß-Protein
Salvatore Pezzella (Class of 2009) Noel D.
Lazo Carlson School of Chemistry and
Biochemistry, Clark University Worcester, MA
01610
INTRODUCTION
RESULTS AND DISCUSSION
Recently, Lazo et al. studied Aß monomer folding
and identified a decapeptide segment of Aß
(Ala21-Glu22-Asp23-Val24-Gly25-Ser26-Asn27-Lys28-G
ly29-Ala30) within which turn formation appears
to nucleate monomer folding. The turn is
stabilized by hydrophobic interactions between
Val-24 and Lys-28 and by long-range electrostatic
interactions between Lys-28 and either Glu-22 or
Asp-23 (Figure 2).
Alzheimers disease affects over 24 million
people throughout the world, and is currently the
most common form of dementia. There is currently
no cure for Alzheimers disease, which is
degenerative and terminal. Alzheimers can begin
many years before it is diagnosed, and in early
stages usually begins with loss of short-term
memory. As the disease matures, symptoms can
include mood swings, anger, confusion and loss of
language, and long-term memory loss. Minor
bodily functions are gradually lost, followed by
major bodily functions and death. Alzheimers
disease is believed to be caused by the formation
of neurotoxic assemblies formed by the amyloid-ß
protein (Aß). Aß is produced from a 770-residue
precursor protein (AßPP) by the action of enzymes
called secretases (Figure 1). The cleavage by
?-secretase occurs in the membrane to produce
Aß40 and Aß42, the two predominant forms of Aß.
The three-dimensional structure of Aß has not
been elucidated as of yet, although the amino
acid sequence has been determined (Figure 1).
Aß40
Aß40
Figure 3 HPLC chromatograms of Aß40 obtained
using UV detection at 214 nm (top) and 275 nm
(bottom). The fraction containing Aß-40 wild type
eluted between 57.0-58.4 minutes. The mass of
the protein was verified through electrospray
ionization mass spectrometry.
Figure 2. Stereo views of the two turn conformers
found in the Ala-21 to Ala-30 region of Ab. In
Family 1, the turn is stabilized in part by the
electrostatic interaction between Glu-22 and
Lys-28. In Family 2, the electrostatic
interaction is between Asp-23 and Lys-28.
Experimental 4342.9 Expected4343.0
We hypothesize that turn destabilization may
facilitate increased aggregation of Aß. If true,
an attractive therapeutic strategy is to design
small molecules that will stabilize the turn. To
understand further how the turn structure in Aß
is stabilized, we mutated Gly-25 to a ß-glycine
residue. The backbone of this residue is longer
by one carbon relative to glycine. We then
compared the biophysical properties of the mutant
Aß40 to wild-type Aß40.
Wild Type Amyloid ß Sequence
Figure 5. CD spectra indicates that ß-Gly25Aß40
aggregates at a significantly faster rate than
Aß40.
MATERIALS AND METHODS
CONCLUSION
Figure 4. Presented above is the electrospray
ionization mass spectrum of ß-Gly25Aß40. The
masses obtained were as expected, and thus verify
that the correct protein was synthesized.
Wild type Aß40 and ß-Gly25Aß40 were
synthesized by standard solid-phase synthesis
methods using an Applied Biosystems 433A peptide
synthesizer. Purification of the product was
performed via reversed-phase HPLC using a Varian
Prostar HPLC system. Circular dichroism (CD)
spectroscopy, based on the differing absorption
of left and right-handed circularly polarized
light, was used to detect the presence of
ß-sheets which indicates the presence of
fibrillar structures.
The initial data suggests that the addition of
one carbon to the skeletal backbone of the Gly-25
results in a significant increase in the rate of
aggregation of Aß40. It is possible that
lengthening the backbone allows for a greater
degree of flexibility, and thus leads to
destabilization of the Val24-Lys28 turn. This
allows for a greater extent of interactions
between proteins, leading to the observed
increased rate of aggregation.
Figure 1. Schematic representation of AßPP. The
top diagram shows the longest variant of AßPP.
The bottom diagram shows the locations of the
cleavage sites of the secretases. Pathogenic
mutations and their names are indicated by the
arrows below the amino acid sequence.
To ascertain the effect of the mutation on
the aggregation properties of Aß40, circular
dichroism was performed over a number of days.
CD spectra for Aß40 and ß-Gly25Aß40 are shown
in Figure 5.