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Wednesday, February 19, 2014
3489-Pos Board B217
Enthalpy-Entropy Compensation and Isoequilbria Implicate Solvation as
the Driving Force for Amino Acid Conformational Propensity
Siobhan Toal1, Daniel Verbaro2, Reinhard Schweitzer-Stenner1.
Chemistry, Drexel University, Philadelphia, PA, USA, 2Biology, Drexel
University, Philadelphia, PA, USA.
The existence of residual structure in intrinsically disordered proteins (IDPs)
raise questions regarding how disorder/order preferences are encoded in the
peptide sequence. In addition to formation of local secondary structure, individual amino acids sample unique and restricted conformational space, contrary to
the predictions of the random coil model. To understand the thermodynamic
driving forces underlying these newly discovered amino acid conformational
biases, we have examined the thermodynamics governing these intrinsic
conformational propensities in a series of host-guest experiments on model
‘‘GxG’’ peptides in aqueous solution. Global two-state pPII/b thermodynamic
analysis of UVCD and HNMR data revealed the existence of a nearly exact
enthalpy-entropy compensation for the pPII-b equilibrium for all residues.
The obtained DH/DS values exhibit a nearly perfect linear relationship reflecting compensation temperature of 295K 5 2 K. We identified iso-equilibria for
two subsets of the investigated peptides, indicating that pPII and b propensities
of most residues become similar at physiological temperature, in spite of rather
large differences between respective enthalpic and entropic differences. Interestingly, alanine, aspartic acid, and threonine, which have largely biased
intrinsic conformational propensities, are the only amino acid residues that
do not share iso-equilibria. We assign the isoequilibria to a common
enthalpy-entropy compensation which occurs in the hydration shell of side
chains with considerable hydrophobic content. Aspartic acid and threonine
deviate from this behavior owing to their capability to form hydrogen bonds
with the backbone even in extended structures, whereas the solvation energy
of alanine is dominated by backbone - solvent interactions.
3490-Pos Board B218
Nearest Neighbor Interactions Attenuate Intrinsic Amino Acid Conformational Preferences: A Combined Vibrational and NMR Study
Siobhan Toal1, Reinhard Schweitzer-Stenner1, Karin Rybka2,
Harold Schwalbe2.
Chemistry, Drexel University, Philadelphia, PA, USA, 2Institute of Organic
Chemistry and Chemical Biology, Johann Wolfgang Goethe University,
Frankfurt, Germany.
Contrary to the isolated pair hypothesis, a pivotal ingredient of the random
coil model, conformational distributions of amino acid residues in unfolded
proteins cannot be considered as isolated from their nearest neighbors. To
enable structural predictions of unfolded proteins or peptides (IDPs) knowledge about intrinsic propensities of amino acid residues must be complemented by information on nearest-neighbor interactions. To explore this
influence, we preformed a joint vibrational and 2D-NMR spectroscopy study
of selected ‘‘GxyG’’ host-guest peptides in aqueous solution: GDyG, GSyG,
GxLG, GxVG, where x/y={A,K,LV}. The choice of D and S (L and V) for
the x (y) position was motivated by their documented ability to drastically
change the distribution of alanine in xAy tripeptides of truncated coil libraries.
The observed spectral information were analyzed in terms of superpositions of
2D-Gaussian functions in the Ramachandran space, representing subensembles of pPII-, b-strand-, helical-, and turn-like conformations. Results
reveal that ‘‘turn forming residues’’ and hydrophobic residues have very
strong effects on the conformation of their nearest neighbors. A direct comparison of the conformational distribution for S and D in GSAG ,GSLG,
and GDAG with corresponding distributions in GxG peptiess reveals that
hydrophobic neighbors cause drastic reductions of turn propensities. As a
neighbor, K eliminates the asx turn propensity of D. In addition, insertion
of these ’turn forming’ residues (S, and D) causes conformational redistributions of L, A, and K from pPII to more extended b strand-like conformations
while at the same time increasing the overall turn content. Concomitantly, the
positions of pPII and particularly of b distributions are shifted to lower c
values. Taken together, our results indicate that Dx and Sx motifs might act
as conformational randomizers in proteins, attenuating intrinsic propensities
of neighboring residues.
3491-Pos Board B219
Electrostatics-Dependent Shape of the Intrinsically-Disordered Protein
Gregory W. Gomes1,2, Baoxu Liu1,2, Patrick Farber3,4, Veronika Csizmok3,4,
Julie Forman-Kay3,4, Claudiu C. Gradinaru1,2.
Department of Physics, University of Toronto, Toronto, ON, Canada,
Department of Chemical and Physical Sciences, University of Toronto
Mississauga, Mississauga, ON, Canada, 3Molecular Structure and Function
Program, Hospital for Sick Children, Toronto, ON, Canada, 4Department of
Biochemistry, University of Toronto, Toronto, ON, Canada.
For intrinsically disordered proteins (IDPs) containing a high density of
charged residues, a Gaussian chain is an inadequate description of the protein’s
shape. One such IDP is Sic1, a highly positively charged IDP in the budding
yeast Saccharomyces Cerevisiae which prevents the cell cycle from entering
the S-phase from the G1-phase. Sic1’s binding affinity for Cdc4 is highly phosphorylation state dependent, although the corresponding physical basis is not
fully understood. NMR data supports the presence of a dynamic complex of
Sic1:Cdc4 and a poly-electrostatic model has been proposed by Forman-Kay
and coworkers.
We studied the Sic1 N-terminal targeting region (1-90) to better understand
the role of intrachain electrostatics, and to compare with the structural ensembles calculated from NMR and SAXS data. Sic1 is characterized using timeresolved fluorescence anisotropy (TRFA), which is sensitive to rotational and
conformational dynamics. Sic1 exists in a dynamic ensemble of conformations and ensemble-averaged experiments have limitations in identifying
and characterizing static and/or dynamic inhomogeneity in the motional dynamics. Therefore, we also performed burst spectroscopy and singlemolecule TRFA on freely diffusing Sic1. Shape and flexibility were probed
by varying the degree of intrachain repulsion screening through adjusting
salt concentrations and described within a polymer physics framework, the
polyelectrolyte model. To investigate the relative contributions of ‘‘global
rotation’’ and ‘‘conformational flexibility’’, Sic1 was labelled at three different sites. Sic1 is found to be well modelled as a prolate ellipsoid with internal flexibility.
The single-molecule derived TRFA distribution data may be valuable as a
constraint incorporated in future conformational ensemble calculations,
complementary to SAXS and NMR data. Additionally, these measurements
raise questions about the accuracy of highly charged IDP’s radii of gyration
when calculated from sm-FRET derived end-to-end distances, which often assume a Gaussian chain model for the end-to-end distance probability
3492-Pos Board B220
Development of Intrinsically Disordered Protein Brushes as Smart Biomaterials
Nithya Srinivasan, Maniraj Bhagawati, Badriprasad Ananthanarayanan,
Sanjay Kumar.
Univ. of California, Berkeley, Berkeley, CA, USA.
The grafting of polymer chains onto surfaces at high density to yield "polymer brush" coatings is a widely employed strategy to reduce biofouling and
interfacial friction. Here we report the design, development, and characterization of synthetic intrinsically disordered proteins (IDPs) based on the
C-terminal sidearm domain of the human neurofilament heavy chain protein.
Dynamic light scattering measurements indicate that the recombinant wild
type protein adopts an extended conformation with a hydrodynamic radius
of ~10 nm, much larger than the expected value for a folded protein.
This macromolecule adopts an extended, disordered conformation in solution
and can be grafted at high density in an oriented fashion to solid supports, as
measured by quartz crystal microbalance with dissipation studies. The
resulting IDP brushes are responsive to multiple stimuli such as solution
pH and ionic strength. The swelling and collapsing behavior of the brush
exhibits similar salt-dependence and comparable dynamic range to a synthetic, weak polyelectrolyte brush. This study provides evidence that
stimuli-responsive polymer brushes can be fabricated from proteins and introduces these molecules as a new class of ‘‘smart’’ biomaterial building