Haydar, Ali

Kerr, Becky

Kumar, T.K.S.

Elsevier - Publisher Connector 

Sunday, March 6, 2011 49a252-Pos Board B52
Rational Design of Human Fibroblast Growth Factor with Enhanced
Stability
Ali Haydar, Becky Kerr, T.K.S. Kumar.
Fibroblast growth factors (FGFs) constitute a large family of heparin-binding
proteins which control the proliferation, differentiation, and migration of a va-
riety of cell types. For its transduction of cellular signals, FGF must bind to the
extracellular domains of fibroblast growth factor receptor (FGFR), a transmem-
brane tyrosine kinase receptor. The resulting FGF-FGFR complex leads to re-
ceptor dimerization which stimulates a phosphorylation cascade within the cell
resulting in a variety of cellular outcomes.
FGF also binds to a second class of receptors for which it has a lower affinity,
heparan sulfate proteoglycans (HSPGs). Heparanase treatment of intact cells
has shown that FGF fails to bind to FGFR resulting in non-proliferation of
the cells, indicating the importance of HSPG-binding. Closely related to hep-
aran sulfate is heparin, a structurally-similar proteoglycan which can bind to
FGF isofunctionally.
The role of heparin in FGF-FGFR interactions has been discussed greatly. Hep-
arin is believed to play a role in FGF stability, protecting it from thermal dena-
turation and proteolytic degradation. It has also been proposed that HSPG will
increase the binding affinity of FGF for FGFR and will stabilize the dimeriza-
tion of FGFR. Some studies have shown, however, that heparin binding may
not be essential to FGF-FGFR binding. The goal of this study was to define
the role of heparin in human FGF-1 (hFGF-1) stability and cell proliferation
activities. A His-tagged quadruple mutant with mutations made in the defined
heparin-binding region of hFGF-1 at the N128K, Y139K, G140R, and Q141K
positions (Quad-His) was created prior to the beginning of this study, and the
mutant was compared against wild type hFGF-1 through a variety of biophys-
ical techniques. Experiments were conducted with both heparin and sucrose oc-
tasulfate (SOS), an analog commonly used as a structural and functional mimic
for heparin.
253-Pos Board B53
Sequence Dependence of Coiled-Coil Formation and Stability Studied
Using a Coarse-Grained Atomistic Model
Sara Sadeghi, Eldon Emberly.
The recurrent coiled-coil domain in proteins consists of multiple helices
that intertwine to form a superhelix. Superhelical formation is driven primarily
by the packing of hydrophobic residues that pattern each helix making up the
coil. An examination of the sequences forming naturally occurring coiled-coils
reveals a high degree of variation, yet all the sequences ultimately fold into left-
handed superhelical structures. We have developed a coarse grained atomistic
folding model for coiled-coil formation that only requires a hydrophobic-polar
pattern for the input sequence[1] and are able to generate coiled-coil structures
that have a low root mean square distance(RMSD) to known structures. We
have considered different parameters in our energy function and tried to opti-
mize them to get lowest RMSD. Besides correctly predicting structure from se-
quence, we have applied the model to study both the thermodynamic and
mechanical stability to sequence mutations for the selected coiled coils. The
model provides insights into key sequence characteristics that aid both stability
and the kinetics of coil formation.
[1] S.Sadeghi & E.Emberly ; Phys. Rev. E.80, 061909 (2009).
254-Pos Board B54
Structural Basis of WHAMM to Regulate Golgi Membrane Transporta-
tion via Interacting with Both Microtubules and Membranes
Qingtao Shen, Kenneth G. Campellone, Charles V. Sindelar,
Matthew D. Welch, Hongwei Wang.
WHAMM protein is a newly identified mammalian nucleation-promoting pro-
tein, and it is comprised of an N-terminal domain that mediates Golgi mem-
brane association, a coiled-coil domain (CC) that binds to microtubule, and
a WCA domain that stimulates Arp2/3-mediated actin polymerization.
WHAMM protein plays key roles in both maintaining Golgi structure and fa-
cilitating anterograde membrane transport. The molecular mechanism for
WHAMM to fulfill its functions remains unknown.We have used cryo-electron
microscopy (cryo-EM) to investigate the structural basis of WHAMM’s inter-
action with microtubules and membranes.
Our in vitro pelleting assay shows that both full-length WHAMM protein and
the CC domain have high binding affinity to Taxol-stabilized microtubules at
around 360 nM. This is verified by Cryo-EM observation that both full-length
WHAMM protein and CC assemble around microtubules within a few min-
utes of incubation. Image analysis of the micrographs indicates that the pro-
teins assemble with stoichiometric binding to the alpha-beta tubulinheterodimers in the microtubule lattice. We performed three-dimensional re-
construction of the WHAMM and CC assemblies around microtubules and re-
vealed that WHAMM binds to the outer surface of microtubules via direct
interaction between its CC domain with tubulin dimer. The assembly of
WHAMM onto microtubule exposes the N-terminal domain ready to bind
membranes.
We have further used fluorescence microscopy to demonstrate that vesicles
made of lipids from liver extract can be recruited to microtubules in the pres-
ence of WHAMM in an in vitro assay. Cryo-EM of such a specimen verified the
cross-bridge between the lipid vesicles and microtubules by WHAMM and
showed that the interaction causes the vesicles to change their shapes along
the microtubules.
255-Pos Board B55
Understanding Functional Tuning, Robustness, and Evolvability in Pro-
teins By High-Throughput Biophysics
Wouter D. Hoff.
Proteins consist of only 20 different amino acids with very modest chemical
reactivity, but perform a breathtaking range of functions. A central question in
biophysics is to understand how proteins achieve such functional versatility. A
key recent advance in understanding protein structure-function relationships is
the robustness of proteins against point mutations, which implies that only
a small subset of residues determines functional properties. We tested this pre-
diction using photoactive yellow protein (PYP), a 125-residue prototype of the
PAS domain superfamily of signaling proteins. PAS domains are defined by
a small number of conserved residues of unknown function. Our high-
throughput biophysical measurements on a complete ala scan of purified
PYP mutants characterizing active site properties, functional kinetics, stabil-
ity, and production level reveal that 124 mutants retain the characteristic pho-
tocycle of PYP, but that the majority of substitutions significantly alter
functional properties (Philip et al., PNAS 2010, early edition 10.1073/
1006660107). Only 35% of substitutions that strongly affect function are lo-
cated at the active site. Unexpectedly, most PAS-conserved residues are re-
quired for maintaining protein production. The photocycle kinetics are
significantly altered by substitutions at 58 positions and span a 3,000-fold
range, allowing us to identify conserved interactions governing allosteric
switching in PYP. The data also shed light on two classic examples of func-
tional tuning of active site groups, shifts in pKa value and shifts in absorbance
maximum for spectral tuning, and result in a novel model for spectral tuning
based on changes in the shape of the energy surfaces involved (Philip et al.,
2010, PNAS 107: 5821-5826). The results show that PYP combines robust-
ness with a high degree of evolvability, imply production level as an impor-
tant factor in protein evolution, and provide new insights into the functional
tuning of active site residues.
256-Pos Board B56
Local Conformational Fluctuations of the Backbone Affect the pKa Values
of Ionizable Groups in Proteins
Brian M. Doctrow, Kelli L. Baran, Jamie L. Schlessman,
Bertrand Garcı´a-Moreno.
The pKa values of all 20 Asp and Glu residues in staphylococcal nuclease
(SNase) have been measured previously using NMR spectroscopy. Some of
these pKa values are shifted relative to the normal values for Asp and Glu in
water, but most are not. This does not agree with structure-based pKa calcula-
tions, according to which most of the pKa values are depressed because of
strong Coulomb interactions with basic residues. This discrepancy persists
even in calculations that treat the protein interior with the dielectric constant
of water, suggesting that the crystal structure does not fully represent the en-
semble average in solution. This idea is supported by calculations showing
that the acid unfolding properties of SNase are reproduced better when the pro-
tein is treated as an ensemble of structures rather than as a static structure. We
have tested the hypothesis that subtle, local conformational fluctuations con-
tribute significantly to the pKa values of ionizable groups. This was examined
experimentally by measuring pKa values of Asp, Glu, and His residues with
NMR spectroscopy in variants of SNase with Gly substitutions at strategic lo-
cations, designed to enhance the flexibility of the backbone. Such substitutions
should affect pKa values without affecting the protein structure. Consistent with
our hypothesis, several Gly substitutions were found to shift the pKa values of
ionizable groups without affecting the structure noticeably. Efforts are under-
way to determine what the effects of Gly substitutions on structure and fluctu-
ations are. Our results suggest that the high apparent polarizability of proteins
might be due to local or subglobal unfolding. We will present a computational
model designed to capture the effects of local unfolding on pKa values in
proteins.


Rational Design of Human Fibroblast Growth Factor with Enhanced Stability 

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Rational Design of Human Fibroblast Growth Factor with Enhanced Stability

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