Sunday, February 26, 2012 55a
Temperature experiments show a two-step unfolding of the protein. Above
45�C we have molten globule kind of loosened structure, and the complete un-
folding occurs above 70�C. The unfolding is accompanied with aggregation of
the protein. The aggregation can however be prevented by relatively low pres-
sure of 0.2 GPa.
Using the above phase transition points the T-p phase diagram of parvalbumin
was determined in presence of Ca2þ. It shows a very complex pattern, contain-
ing two different molten globule phases besides the native, unfolded and aggre-
gated ones.

274-Pos Board B60
Protein Conformational Intermediates Characterized with Novel High
Pressure EPR and CD Techniques
John McCoy, Joseph Horwitz, Wayne L. Hubbell.
UCLA, Los Angeles, CA, USA.
Regulation of protein function is often linked to conformational intermediates
that exist in equilibrium with the ground state. In many cases, these intermedi-
ate states exist as only a small fraction of the total protein conformational en-
semble. It has been shown that high hydrostatic pressure is an invaluable tool
for populating low-lying, excited states to levels amenable to spectroscopic de-
tection. Here we demonstrate the usefulness of high pressure for populating
such states as monitored by two novel techniques: High pressure electron para-
magnetic resonance spectroscopy (EPR) and high pressure circular dichroism
(CD). High pressure EPR was used in conjunction with site-directed spin label-
ing to monitor changes in local protein structure with applied pressure (up to
400 MPa). The relative partial molar volume and isothermal compressibility
of each conformational substate was determined from the pressure dependence
of the equilibrium constant determined from the continuous wave EPR spectra.
High pressure CD was employed to detect global changes in secondary and ter-
tiary structure at elevated pressure (up to 200 MPa). The combination of site-
specific and global information provided by these techniques provides a more
complete description of pressure excited intermediate states. Data from apo-
myoglobin and a cavity containing mutant (L99A) of T4 lysozyme are
presented.

275-Pos Board B61
Volumetric Characterization of Interactions of Glycine Betaine with
Protein Groups
Yuen Lai Shek, Tigran V. Chalikian.
University of Toronto, Toronto, ON, Canada.
We report the partial molar volumes and adiabatic compressibilities of N-acetyl
amino acid amides and oligoglycines at glycine betaine (GB) concentrations
ranging from 0 to 4 M. We use these results to evaluate the volumetric contri-
butions of amino acid side chains and the glycyl unit (–CH2CONH–) as a func-
tion of GB concentration. We analyze the resulting GB dependences within the
framework of a statistical thermodynamic model and evaluate the equilibrium
constant for the reaction in which a GB molecule binds each of the functional-
ities under study replacing four water molecules. We calculate the free energy
of the transfer of functional groups from water to concentrated GB solutions,
DGtr, as the sum of a change in the free energy of cavity formation, DDGC,
and the differential free energy of solute-solvent interactions, DDGI, in a con-
centrated GB solution and water. Our results suggest that the transfer free en-
ergy, DGtr, results from a fine balance between the large DDGC and DDGI

contributions. The range of the magnitudes and the shape of the GB dependence
of DGtr depend on the identity of a specific solute group. The interplay between
DDGC and DDGI results in pronounced maxima in the GB dependences of DGtr

for the Val, Leu, Ile, Trp, Tyr, and Gln side chains as well as the glycyl unit.
This observation is in qualitative agreement with the experimental maxima
in the TM-versus-GB concentration plots reported for ribonuclease A and
lysozyme.

276-Pos Board B62
Protein Stability and Macromolecular Crowding
Mohona Sarkar, William B. Monteith, Yaqiang Wang, Gary J. Pielak.
UNC Chapel Hill, Chapel Hill, NC, USA.
The environment inside cells is vastly different from the dilute, idealized con-
ditions used in nearly all biophysical studies. Cells are exceptionally complex
and contain macromolecules at concentrations exceeding 300 g/L. There are at
least two profound consequences of the cellular environment that impact glob-
ular proteins. First, cells are highly ‘‘crowded’’. The resulting decrease in avail-
able volume should increase protein’s stability. Second, because the bulk
concentration of protein functional groups in cells is so high, nonspecific inter-
actions between a protein and other cellular components, even if weak individ-
ually, can sum to large net, typically destabilizing, effects. in other words,
crowding is a battle between the excluded volume effects and nonspecific inter-
actions. We aim to identify the winner in different crowding environments,
starting with inert synthetic polymers, moving to homogeneous protein crow-
ders and ultimately to the endogenous cellular components of bacteria. We
have already shown that excluded volume plays a predominant role for syn-
thetic polymers, but nonspecific interactions with the test protein are stronger
for more biologically relevant crowders.

277-Pos Board B63
Direct Quantification of the Effects of Osmolytes on Protein Unfolding and
Refolding Pathways using AFM Techniques
Paul Bujalowski, Liang Ma, Andres Oberhauser.
UTMB, Galveston, TX, USA.
There is considerable interest in developing new effective therapies based
on small molecules for the treatment of devastating pathologies, such as
Alzheimer, Huntington and Parkinson diseases. Osmolytes are a class of small
organic molecules found in all taxa that can profoundly affect protein stability
and aggregation. Osmolytes such as sorbitol or trimethylamine-N- oxide can
act as ‘‘chemical chaperones’’ by increasing the stability of native proteins, as-
sisting refolding of unfolded polypeptides, inhibiting protein aggregation.
However, the mechanism of protecting osmolytes action on protein folding
and stability remains controversial, where the protein backbone seems to
play a key role. Here we used single-molecule AFM to systematically analyze
the effect of several naturally occurring osmolytes (sorbitol, sarcosine, TMAO,
inositol, trehalose, proline, glycerol, and taurine) on the stability and folding
kinetics on PKD domains which are normally exposed to high urea concentra-
tions in the kidney. Our approach enables us to directly quantify the effects of
osmolytes on the folded state and on the exposed protein backbone in the
absence of chemical denaturants. Here we show that a mixture of different pro-
tecting osmolytes (e.g. 0.5M sorbitol þ 0.5M sarcosine) counteracts the effects
of 1M urea equally well as 1M sorbitol on the unfolding/refolding rates. These
results indicate an additive effect of these protecting osmolytes on domain sta-
bility. However, we also found osmolytes mixtures that work synergistically on
protein stability. Our results demonstrate a robust approach to study the mech-
anism of action of osmolytes at the single protein level.

278-Pos Board B64
Developing Solutes as Probes of Protein and DNA Processes
Emily J. Guinn, Laurel M. Pegram, Michael W. Capp, Michelle N. Pollock,
Hyo Cha, M.T. Record.
UW-Madison, Madison, WI, USA.
Solutes have a broad range of effects on biopolymer processes, forming a spec-
trum from destabilizers like urea to more stabilizing osmolytes like proline,
glycine betaine (GB) and KGlutamate to secondary structure inducers like tri-
fluorethanol. To explain these effects and develop these solutes as probes of
interface formation and large scale conformational changes in protein and
DNA processes, we quantify the thermodynamics of their competition with
water to interact with different types of biopolymer surface (e.g. aliphatic
and aromatic C, polar and charged O and N) using model compounds display-
ing one or more surface type. Preferential interactions between the solutes and
model compounds relative to their interactions with water are determined by
osmometry or solubility and dissected using a novel coarse-grained analysis
to obtain interaction potentials quantifying the solute’s interaction with each
significant type of biopolymer surface. Microscopic local-bulk partition coeffi-
cients Kp for the accumulation or exclusion of the solute in the water of hydra-
tion of these surfaces relative to bulk water are obtained. We used model
compounds representing protein surface types to obtain Kp values for urea
and GB, revealing that urea accumulates moderately at amide O and weakly
at aliphatic C, while GB is excluded from both. These results provide both ther-
modynamic and molecular explanations for the opposite effects of urea and GB
on protein stability, as well as deductions about strengths of amide NH - amide
O and amide NH - amide N hydrogen bonds relative to hydrogen bonds to
water. Urea and GB m-values for protein folding and other protein processes
are interpreted and predicted using these interaction potentials or Kp values.
We also determine interactions of these solutes with nucleic acid surface types
to develop the ability to probe protein-nucleic acid interactions.
Supported by NIH GM47022.

279-Pos Board B65
Protein Hydration and Excluded Volume Interactions in Protein Folding
and Stability: On the Mechanism of Protein Stabilization by Osmolytes
Marcio F. Colombo, Alexandre C.D Neves, Sandra R. Marão,
Jorge Chahine.
Universidade Estadual Paulista, São José do Rio Preto, Brazil.
Several osmolytes ranging in size from ~90-1600 cm3.mol-1 are shown to sta-
bilize the folded states of apo-Mb at pH denaturing conditions. The action of



56a Sunday, February 26, 2012
these solutes is consistent with an osmotic stress effect, i.e., with their effect on
the chemical potential of water modulating the equilibrium between folded and
unfolded states via protein differential hydration. Moreover, the free energy of
protein folding depends on solute size. We show that the apparent difference in
hydration between molten globule and unfolded states of apo-Mb increases lin-
early with increasing solute sizes, while the free energy change of protein hy-
dration upon folding decreases with the inverse of molar solute apparent
volume.We have analyzed these size effects considering the contribution of ex-
cluded volume interactions to protein folding via Monte Carlo simulations of
a self-avoiding walk chain in a cubic lattice with hardcore solutes. It is shown
that solute-chain self-avoiding interaction decrease the conformational entropy
of the chain in proportion to its square radius of gyration, and to the solute vol-
ume fraction occupied by the solute. The computational results translate into
a difference in the chemical potential between folded and unfolded protein
states that remarkably predicts the experimental influence of solute sizes and
water chemical potential on the free energy change of apo-Mb refolding in-
duced by osmolytes. Thus, this work may stablish a quantitative link between
protein hydration and protein excluded-volume interactions and their effect on
the energetic of protein folding.

280-Pos Board B66
A Partially Structured Molten Globule Protein
Joerg Reichenwallner, Mohammed Chakour, Wolfgang E. Trommer.
Kaiserslautern Technical University, Kaiserslautern, Germany.
Maltose binding protein (MBP) from E. coliwas shown to bind maltose even in
its molten globule state, although with substantially reduced affinity. The native
protein of which the X-ray structure is known, is devoid of cysteines. Seven dif-
ferent mutants with two cysteines each were labeled with the MTS-SL. Dis-
tances from the active site as derived from the X-ray structure vary from 14
to 31 Å. DEER measurements have so far shown very good agreement between
the X-ray data and the native structure. Here we compare distances in the native
protein with those in the molten globule state.

281-Pos Board B67
Collagen Unfolding Determines Fluid Transfer in the Interstitial Matrix
Maria P. McGee, Michael Morykwas, Louis Argenta.
Wake-Forest University Medical School, Winston-salem, NC, USA.
Local unfolding of collagen at physiologic temperatures is thought to facilitate
interactions with enzymes and scaffold molecules during inflammation, tissue
remodeling, and wound healing. Previous data showing high interstitial hydra-
tion potential (HP) in human and porcine dermis after collagen thermal unfold-
ing and fibroblast death suggest that it also plays a role in local modulation of
interstitial flows. To test this hypothesis, collagen was progressively unfolded
in situ, and changes in HP and water influx-rate within the matrix were
measured as a function of the extent of unfolding, which was quantified by dif-
ferential scanning calorimetry in full-thickness dermal samples after timed
heat-treatment at 60�C and equilibration at 4�C. HP was determined by os-
motic stress techniques, and influx-rates from time-dependent gravimetric
changes under 35mmHg osmotic counterpressure. Both increased linearly
with the proportion of unfolded collagen: the HP by 1.08 5 0.16 mmHg,
and the influx-rate by 3.19 5 0.39 ml/min /100g per each 1% of collagen un-
folded (R2= 0.93 and 0. 95, respectively). The relative humidity and intensity
of T2-weighed magnetic resonance images of the dermis also increased with
the extent of collagen unfolding, confirming interfacial energy contributions
to the HP - as predicted by the Kelvin relationship - and the expected hydropho-
bic nature of the newly formed protein/water interfaces, respectively. These re-
sults are fully consistent with the hypothesis and point to yet another potentially
important function of local collagen unfolding in tissue homeostasis. As a plau-
sible mechanism for HP and influx-rate increases with collagen unfolding, we
propose that the surface tension of vapor/water interfaces under exposed hydro-
phobic clusters is higher than at hydrophilic interfaces; at nanometer scales,
these differences generate local surface-tension gradients in the matrix that
accelerate water influx and shift the HP.

282-Pos Board B68
Temperature Dependence of Protein Folding in Live Cell
Minghao Guo, Martin Gruebele.
University of Illinois - Urbana Champaign, Urbana, IL, USA.
Protein folding kinetics is known to be non-Arrhenius temperature dependent.
We use Fast Relaxation Imaging (FReI) to measure stability and folding kinet-
ics of FRET-labeled destabilized phosphoglycerate kinase (PGK). With modu-
lated heating laser, we are able to measure the thermodynamics of PGK rapidly
across the midpoint of transition of protein unfolding to minimize baseline
shifts coming from photobleaching and protein aggregation.
We have measured PGK folding kinetics from 295K to 320K both in vitro and
in vivo. Kinetics of PGK as mutiplestate folder can be fitted to stretched expo-
nential. Folding rate and folding mechanism of PGK are correlated and both are
strongly dependent on temperature, which can be explained by solvent viscos-
ity and hydrophobic interactions.

283-Pos Board B69
Folding Mechanism of a Precursor Protein of a Peptide Hormone
Mediated by an Intra-Molecular Chaperone
Masaki Okumura1, Yu-ichiro Yoshida2, Hiroshi Yamaguchi1, Yuji Hidaka2.
1Kwansei Gakuin univ., Sanda, Japan, 2Kinki univ., Higashi-osaka, Japan.
Prouroguanylin is a precursor of uroguanylin. The mature form of uroguanylin
contains intra-molecular disulfide bonds (Cys74-Cys82 and Cys77-Cys85). The
propeptide region functions as an intra-molecular chaperone in the formation of
the native conformation and the disulfide pairings of uroguanylin. To elucidate
the mechanism of the propeptide-mediated folding, the pathway associated
with the disulfide-coupled folding of prouroguanylin was examined in detail.
Prouroguanylin, when prepared using an E. coli expression system, was ob-
tained as an inclusion body. Therefore, it was purified as a reduced/denatured
protein by reversed-phase HPLC after solubilization in urea. The folding reac-
tion was carried out 0.1 M Tris/HCl (pH 8.0) at various concentrations of glu-
tathione in the presence and absence of protein disulfide isomerase which
catalyzes the disulfide exchange reaction.
Kinetic analyses of the oxidative folding revealed that two types of intermedi-
ates containing mis-bridged disulfide bonds (namely, isomers 1 and 2 in which
the disulfide bonds were between Cys74-Cys85 and Cys77-Cys82 and Cys74-
Cys77 and Cys82-Cys85 in the mature region, respectively) are predominantly
included in the folding. However, only one type of intermediate containing mis-
disulfide bonds, isomer 2, was able to proceed to the native conformation of
prouroguanylin, regardless of the presence of protein disulfide isomerase.
The results of these experiments will be discussed in this presentation.

284-Pos Board B70
Role of Leu66 in the Folding of Uroguanylin Assisted by Intra-Molecular
Chaperone
Yu-ichiro Yoshida1, Masaki Okumura2, Shigeru Shimamoto1,
Hiroshi Yamaguchi2, Yuji Hidaka1.
1Kinki University, Higashi-Osaka, Japan, 2Kwansei Gakuin University,
Sanda, Japan.
Uroguanylin is maturated via the processing of a precursor protein, prourogua-
nylin. The pro-peptide region of the precursor protein of uroguanylin regulates
the formation of the native structure of uroguanylin, by serving as an intra-
molecular chaperone. To estimate the role of the individual amino acid residues
of the pro-peptide region in chaperon function, we previously prepared Gly or
Ala mutants and the folding of the mutant proteins were examined. The results
revealed that, except for Cys residues, only the Leu66 residue critically affected
the folding of the mature region, uroguanylin. To further investigate the role of
the Leu66 residue in the folding of uroguanylin, it was mutated to several dif-
ferent amino acid residues, such as Gly, Ala, Val, and Ile.
The cDNA’s encoding the mutant proteins were amplified by polymerase chain
reaction and inserted into pET17b vector. The mutant proteins were expressed
using the T7-promoter expression system in E. coli BL21(DE3) cells. The mu-
tant proteins were obtained as inclusion bodies and solubilized in 0.1 M Tris/
HCl (pH 8.0) containing 8 M urea and dithiothreitol. The reduced forms of
the mutant proteins were purified by reversed-phase high performance liquid
chromatography (HPLC) and identified by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry analyses.
The oxidative folding of the mutant proteins was carried out in the presence
of reduced and oxidized forms of glutathione and the progress monitored by
HPLC.
The results of these experiments will be discussed in this paper.

285-Pos Board B71
Evaluating a Key Player in Acute Heart Failure: Interaction Surfaces
and Structural Details of Interleukin-33
Kaitlin Fisher.
UCSD, San Diego, CA, USA.
The newest member of the Interleukin-1 family of proteins is IL-33. IL-33 was
recently discovered in 2005 and since then has been identified as a key partic-
ipant in immune and inflammatory responses through association with the IL-1
receptor family member ST2. However, the structural homology between IL33
and other members of the interleukin family are low- presenting unique se-
quence identity, unique receptor interactions, and potentially unique signaling
mechanisms associated with its activity. The current, limited understanding of


	Protein Conformational Intermediates Characterized with Novel High Pressure EPR and CD Techniques
	Volumetric Characterization of Interactions of Glycine Betaine with Protein Groups
	Protein Stability and Macromolecular Crowding
	Direct Quantification of the Effects of Osmolytes on Protein Unfolding and Refolding Pathways using AFM Techniques
	Developing Solutes as Probes of Protein and DNA Processes
	Protein Hydration and Excluded Volume Interactions in Protein Folding and Stability: On the Mechanism of Protein Stabilizat ...
	A Partially Structured Molten Globule Protein
	Collagen Unfolding Determines Fluid Transfer in the Interstitial Matrix
	Temperature Dependence of Protein Folding in Live Cell
	Folding Mechanism of a Precursor Protein of a Peptide Hormone Mediated by an Intra-Molecular Chaperone
	Role of Leu66 in the Folding of Uroguanylin Assisted by Intra-Molecular Chaperone
	Evaluating a Key Player in Acute Heart Failure: Interaction Surfaces and Structural Details of Interleukin-33