# Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II fromNatronomonas pharaonis

Authors: Ivan L. Budyak; Olga S. Mironova; Naveena Yanamala; Vijayalaxmi Manoharan; Georg Büldt; Ramona Schlesinger; Judith Klein-Seetharaman
Journal: Journal of Biophysics
 (2008)

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## Abstract

Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt) from the halophilic archaeonNatronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved. Here, we conducted CD, FTIR, and NMR spectroscopic studies under different conditions to address this question. We provide evidence that pHtrII-cyt is highly dynamic with strong helical propensity, which allows it to change from monomeric to dimeric helical coiled-coil states without undergoing dramatic shape changes. A statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl-accepting chemotaxis protein family.


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## Body

## 1. Introduction

Response to external stimuli
is important for all living organisms. Prokaryotes and lower eukaryotes are
able to move towards favorable and away from unfavorable conditions including
chemicals and light, referred to as chemotaxis and phototaxis, respectively [1, 2]. Repellent phototaxis in the halophilic archaeon Natronomonas pharaonis (also known as Natronobacterium pharaonis) is initiated by light activation of
sensory rhodopsin II (pSRII), a 7 α-helical membrane receptor related to
bacteriorhodopsin [3]. Activation of pSRII induces structural changes in its cognate
transducer protein, pHtrII, a 2 α-helical membrane protein with a long
cytoplasmic extension that transmits the signal further to induce subsequent
phosphorylation processes that control flagellar motion [2]. The crystal structures of pSRII alone [4, 5] and in complex with the transmembrane domain of pHtrII (residues 1–114,
electron density was observed for residues 22–82) have been recently solved,
both in the inactive dark state and in the light-activated conformation [6, 7]. A peptide fragment corresponding to the juxtamembrane residues 83–149
of the above protein was shown by NMR spectroscopy to be unstructured in
solution, but to undergo a structural transition upon interaction with pSRII [8]. EPR spectroscopy of the N-terminal pHtrII protein fragment (residues
1–157) also showed that the juxtamembrane domain has molten globule-like characteristics
[9]. However, the structure of the long cytoplasmic domain of pHtrII
(pHtrII-cyt), which directly interacts with the proteins of the signaling
cascade, remains unknown.pHtrII-cyt exhibits
sequence homology to the cytoplasmic domains of receptors in eubacteria
including the well-studiedEscherichia
coli serine (Tsr-cyt) and aspartate (Tar-cyt) receptors [10]. The identity between pHtrII-cyt and the corresponding sequence of
Tsr-cyt is 24%. For comparison, the overall identity between Tsr-cyt and
Tar-cyt is 71%. These sequences belong to the methyl-accepting chemotaxis protein
(MCP) family, which currently (as of March 2008) has more than 5000 identified
members [11]. The functional significance of the homology between phototaxis
transducer proteins and chemoreceptors has been proven through studies of
chimera [12].Tsr-cyt has been
crystallized and its crystal structure corresponds to a helical coiled-coil dimer
[13]. A comparison of the crystal structure of the methylated with the structure of the non-methylated signaling state revealed little changes in the structure, but
significant differences in B-factor values [14] supporting a model, in which receptor activity is determined by the
flexibility of the cytoplasmic domain [15]. Furthermore, the helical Tar-cyt fragment is easily denatured
thermally [16, 17] and has the characteristics of a molten globule [18]. NMR amide-exchange data revealed the presence of a small (10%) core of
slowly exchanging residues, which presumably are involved in tertiary packing
contacts [18]. The size of the core increased as a result of mutation or pH change [16, 17, 19].We have previously shown that
pHtrII-cyt assumes a shape consistent with the Tsr-cyt helical coiled-coil
dimer structure, but CD spectra lacked evidence for a predominantly helical
conformation [20]. Here, we present an in-depth analysis of pHtrII-cyt using CD,
Fourier-transform IR (FTIR), NMR spectroscopy, and statistical sequence
analysis. From these studies, a picture emerges in which pHtrII-cyt has a high
propensity for helical coiled-coil structure, but maintains flexibility to such
an extent that CD is not able to reliably detect it.


## 2. Materials and Methods

### 2.1. Materials

All sources of materials
including preparation of pHtrII-cyt were as described previously [20], unless otherwise stated. Buffers used were phosphate-buffered saline,
0.9 mM CaCl2, 0.5 mM MgCl2, 2.7 mM KCl, 138 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4 (PBS); 10 mM NaPi pH 6.0 (buffer A); 10 mM Tris-HCl pH 8.0 (buffer B); 10 mM
Tris-HCl pH 8.0 in 99%D2O (buffer C); 10 mM NaPi pH 6.0,
10%D2O (buffer D).


### 2.2. CD Spectroscopy

CD spectra were acquired using a Jasco 810 spectropolarimeter (Jasco Inc., Easton,
Md, USA) as described previously [20]. Experimental data θexp were recorded in a 0.2-cm quartz cell at pHtrII-cyt concentration of 0.1 mg/mL and
then converted to the mean residue ellipticity according to [θ]MRW=θexp/(10*l*c*n) where l is the path length
of the cell in cm, c is the protein concentration in M, and n is the number of
residues. [θ]MRW was used as the input data for the CONTIN/LL secondary structure prediction
program from the CDPro software package [21, 22]. The dataset including reference spectra of denaturated proteins
(SDP48) was chosen as the reference one [23]. The error bars represent the standard deviation of values predicted by
CONTIN/LL assuming 10% error in the determination of concentration. The values
obtained from deconvolution should be considered approximate because of inherent
difficulties in CD spectra deconvolution [24, 25] and the known effects of dynamics on reliability of secondary structure
prediction from CD data [26]. However, we use them here to allow quantitative comparison of
structure and dynamics of pHtrII-cyt under different solvent and temperature
conditions.


### 2.3. NMR Spectroscopy

Measurements were performed at +20C° unless stated otherwise. Solutions for NMR
spectroscopic analysis contained 5 mg/mL uniformly N15-labeled
pHtrII-cyt in buffer D. H1-N15-HSQC, longitudinal (T1)
and transverse (T2) relaxation data, and H1-N15
heteronuclear NOE (hetNOE) data of pHtrII-cyt in the absence and presence of
4 M salt were recorded  at +10C° and +20C°
using a Bruker 600 MHz spectrophotometer. Standard Bruker sequences were used to
record H1-, H1-N15-HSQC, and het-NOE spectra. The T1,T2 spectra were recorded using
the pulse sequence schemes described previously [27]. We used a 90° pulse of 50 microseconds for N15 for all the experiments and a 90° pulse of 14 microseconds and 9.6 microseconds for the H1 in 4M salt and water samples,
respectively. The R1(1/T1) and R2(1/T2)
rates of the 4 M salt sample were measured using the relaxation delays of 40,
80, 160, 320, 480, 640 milliseconds and 30, 64, 96, 128, 192, 240 milliseconds,
respectively. Further, the water relaxation rate values were obtained at
relaxation delays of 32, 64, 128, 256, 384, 512 milliseconds for R1 and 32, 64, 128, 192, 256, 320 milliseconds for the R2. HetNOE experiments
were obtained by collecting the spectra with and without H1
presaturation. A proton presaturation period of 3 seconds was applied before
acquiring each H1‐N15 HSQC spectra. The data recorded
were processed using the NMRPipe, NMRview, and Sparky software packages for
further analysis. The R1 and R2 rates were estimated by
fitting the peak intensities at different relaxation delays using the
two-parameter exponential decay function in the numerical recipes package [27, 28]. The hetNOE values for each
peak were estimated by finding the ratios between the peak intensities of the H1-N15 HSQC spectra with and without
proton presaturation [27].


### 2.4. FTIR Spectroscopy

FTIR absorbance spectra were acquired at room temperature using a Bruker Vector
22 IR-spectrometer equipped with a DTGS detector. For film preparation, 150μg of
pHtrII-cyt in buffer B were dried under N2 gas flow and then sealed.
For measurements in solution, pHtrII-cyt was concentrated to 10 mg/mL, and buffer
B was exchanged to buffer C to reduce background absorbance in the amide I
region. 256 scans were taken for each sample at 1cm−1 resolution.
The spectrum of residual water vapor was measured with an empty chamber and
then subtracted manually from the protein spectra.


### 2.5. Sequence-Based
Predictions of Secondary and Coiled-Coil Structure

Sequence-based
secondary structure predictions were performed using all of the prediction
programs (SOPM, SOPMA, HNN, MLRC, DPM, DSC, GOR I, GOR III, GOR IV, PHD,
PREDATOR, and SIMPA96) available on the NPS@web server [29]. The predictions of coiled-coil regions were done by the COILS
prediction server [30] using the recommended window of 28 residues, MTK matrix (derived from
the sequences of myosins, tropomyosins, and keratins). Since coiled-coils are
often fibrous, solvent-exposed structures, all residues except for the internal a and d have a high probability of being hydrophilic. Giving equal
weights to all positions therefore means being biased towards hydrophilic,
charge-rich sequences. To avoid highly charged false positives a 2.5-fold
weighting of positions a, d was used.


### 2.6. Sequence-Based
Predictions of Disorder Propensity

Sequence-based predictions of the dynamic properties
of individual proteins were obtained from the Predictors of Natural Disordered
Regions (PONDR) prediction server [31]. The analysis of the disorder propensities of the MCP signal family was
carried out using RONN disorder prediction software [32, 33].


## 2.1. Materials

All sources of materials
including preparation of pHtrII-cyt were as described previously [20], unless otherwise stated. Buffers used were phosphate-buffered saline,
0.9 mM CaCl2, 0.5 mM MgCl2, 2.7 mM KCl, 138 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4 (PBS); 10 mM NaPi pH 6.0 (buffer A); 10 mM Tris-HCl pH 8.0 (buffer B); 10 mM
Tris-HCl pH 8.0 in 99%D2O (buffer C); 10 mM NaPi pH 6.0,
10%D2O (buffer D).


## 2.2. CD Spectroscopy

CD spectra were acquired using a Jasco 810 spectropolarimeter (Jasco Inc., Easton,
Md, USA) as described previously [20]. Experimental data θexp were recorded in a 0.2-cm quartz cell at pHtrII-cyt concentration of 0.1 mg/mL and
then converted to the mean residue ellipticity according to [θ]MRW=θexp/(10*l*c*n) where l is the path length
of the cell in cm, c is the protein concentration in M, and n is the number of
residues. [θ]MRW was used as the input data for the CONTIN/LL secondary structure prediction
program from the CDPro software package [21, 22]. The dataset including reference spectra of denaturated proteins
(SDP48) was chosen as the reference one [23]. The error bars represent the standard deviation of values predicted by
CONTIN/LL assuming 10% error in the determination of concentration. The values
obtained from deconvolution should be considered approximate because of inherent
difficulties in CD spectra deconvolution [24, 25] and the known effects of dynamics on reliability of secondary structure
prediction from CD data [26]. However, we use them here to allow quantitative comparison of
structure and dynamics of pHtrII-cyt under different solvent and temperature
conditions.


## 2.3. NMR Spectroscopy

Measurements were performed at +20C° unless stated otherwise. Solutions for NMR
spectroscopic analysis contained 5 mg/mL uniformly N15-labeled
pHtrII-cyt in buffer D. H1-N15-HSQC, longitudinal (T1)
and transverse (T2) relaxation data, and H1-N15
heteronuclear NOE (hetNOE) data of pHtrII-cyt in the absence and presence of
4 M salt were recorded  at +10C° and +20C°
using a Bruker 600 MHz spectrophotometer. Standard Bruker sequences were used to
record H1-, H1-N15-HSQC, and het-NOE spectra. The T1,T2 spectra were recorded using
the pulse sequence schemes described previously [27]. We used a 90° pulse of 50 microseconds for N15 for all the experiments and a 90° pulse of 14 microseconds and 9.6 microseconds for the H1 in 4M salt and water samples,
respectively. The R1(1/T1) and R2(1/T2)
rates of the 4 M salt sample were measured using the relaxation delays of 40,
80, 160, 320, 480, 640 milliseconds and 30, 64, 96, 128, 192, 240 milliseconds,
respectively. Further, the water relaxation rate values were obtained at
relaxation delays of 32, 64, 128, 256, 384, 512 milliseconds for R1 and 32, 64, 128, 192, 256, 320 milliseconds for the R2. HetNOE experiments
were obtained by collecting the spectra with and without H1
presaturation. A proton presaturation period of 3 seconds was applied before
acquiring each H1‐N15 HSQC spectra. The data recorded
were processed using the NMRPipe, NMRview, and Sparky software packages for
further analysis. The R1 and R2 rates were estimated by
fitting the peak intensities at different relaxation delays using the
two-parameter exponential decay function in the numerical recipes package [27, 28]. The hetNOE values for each
peak were estimated by finding the ratios between the peak intensities of the H1-N15 HSQC spectra with and without
proton presaturation [27].


## 2.4. FTIR Spectroscopy

FTIR absorbance spectra were acquired at room temperature using a Bruker Vector
22 IR-spectrometer equipped with a DTGS detector. For film preparation, 150μg of
pHtrII-cyt in buffer B were dried under N2 gas flow and then sealed.
For measurements in solution, pHtrII-cyt was concentrated to 10 mg/mL, and buffer
B was exchanged to buffer C to reduce background absorbance in the amide I
region. 256 scans were taken for each sample at 1cm−1 resolution.
The spectrum of residual water vapor was measured with an empty chamber and
then subtracted manually from the protein spectra.


## 2.5. Sequence-Based
Predictions of Secondary and Coiled-Coil Structure

Sequence-based
secondary structure predictions were performed using all of the prediction
programs (SOPM, SOPMA, HNN, MLRC, DPM, DSC, GOR I, GOR III, GOR IV, PHD,
PREDATOR, and SIMPA96) available on the NPS@web server [29]. The predictions of coiled-coil regions were done by the COILS
prediction server [30] using the recommended window of 28 residues, MTK matrix (derived from
the sequences of myosins, tropomyosins, and keratins). Since coiled-coils are
often fibrous, solvent-exposed structures, all residues except for the internal a and d have a high probability of being hydrophilic. Giving equal
weights to all positions therefore means being biased towards hydrophilic,
charge-rich sequences. To avoid highly charged false positives a 2.5-fold
weighting of positions a, d was used.


## 2.6. Sequence-Based
Predictions of Disorder Propensity

Sequence-based predictions of the dynamic properties
of individual proteins were obtained from the Predictors of Natural Disordered
Regions (PONDR) prediction server [31]. The analysis of the disorder propensities of the MCP signal family was
carried out using RONN disorder prediction software [32, 33].


## 3. Results

### 3.1. pHtrII-cyt
has Strong Propensity for Helix

pHtrII-cyt is predicted to be
a helical coiled-coil based on homology to the serine receptor (Figure1(a)),
high coiled-coil structure propensity (Figure 1(b), top panel), and high helix
propensity with 63% to 97%α-helix content (Figure 1(b),
bottom panel), depending on the prediction algorithm used (see Section 2.5).
These predictions are not consistent with the CD spectra recorded in the
commonly used buffers, PBS, buffer A and buffer B, which were all
indistinguishable and essentially lacked negative ellipticity at 222 nm
characteristic for helix [20]. The values obtained from deconvolution of the spectra were ∼77% unstructured,
∼7% turn, ∼13%β-sheet,
and ∼3%α-helix.
However, control experiments with helix-inducing solvent 2,2,2-trifluoroethanol
(TFE) showed a gradual rise in negative ellipticity at 222 nm with increasing
amounts of TFE added (Figure 1(d)), experimentally validating the high helix
propensity inferred from the pHtrII-cyt sequence.(a) X-ray crystal
structure model of the Tsr-cyt (PDB ID 1QU7) corresponding to identical
positions of the pHtrII-cyt investigated in this study. (b) Top, prediction of
coiled-coil structure for pHtrII-cyt by the COILS software (Section2.5)
and below, prediction of secondary structure for pHtrII-cyt by the DSC software
(Section 2.5). (c) Comparison of FTIR spectra of 10 mg/mL pHtrII-cyt in
buffer C (solid line) and in dry film (dashed line) at room temperature. (d)–(f) Effects
of increasing (d) TFE, (e) glycerol (v/v),
and (f) ammonium sulfate (in % of saturation) concentrations on far-UV CD
spectra of pHtrII-cyt recorded at protein concentration of 0.1 mg/mL at +20C°
(left panel, colors correspond to TFE, glycerol, or ammonium sulfate
concentration) and deduced secondary structure content (right panel, colors
correspond to the content of secondary structure elements). The secondary
structure content estimations were generated by the CONTIN/LL program from the
CDPro package (Section 2.5); secondary structure elements are designated
as follows: helix—black, sheet—red, turn—green, and random coil—blue.
(a)(b)(c)(d)(e)(f)


### 3.2. Dehydration
Stabilizes Helix Structure

Figure1(c) shows FTIR spectra
in the amide I region for pHtrII-cyt in buffer C (solid line) and in dry film
(dashed line). The position of the maximum of the amide I band in buffer C at
1645cm−1 is characteristic of a disordered conformation. In dry
film, a shift to 1654cm−1 was observed, a value that is
characteristic of α-helical secondary structure [25, 34].


### 3.3. Small
Molecule Osmolytes Stabilize Helix Structure

Addition of glycerol to PBS
at concentrations of 60%(v/v) and
higher greatly stabilized helix content which based on spectral deconvolution
rose from ∼19% in 60%(v/v) glycerol
to ∼59% in 90%(v/v) glycerol. β-sheet and turn contributed,
respectively, ∼5% and ∼12% to the spectrum in 90%(v/v) glycerol (Figure 1(e)). In sucrose, at 90% (w/v) saturation, α-helix
content was ∼37%, where β-sheet and turn contributed,
respectively, ∼10% and ∼21%, to the spectrum (data not shown).


### 3.4. Salts
Affect pHtrII-cyt Differentially

Ammonium sulfate, a common
agent to precipitate, stabilize, or facilitate refolding of proteins, up to 30%
saturation, did not significantly alter the CD spectra of pHtrII-cyt obtained
in PBS alone (Figure1(f)). Between 30% and 60%, pHtrII-cyt adopted approximately
50–56%α-helical conformation, while at above 60% saturation, helical content
was reduced and that of random coil increased. Previous crosslinking and
analytical gel-filtration experiments showed that the structural transition was
accompanied by protein aggregation at high ammonium sulfate concentrations [20]. Since the native
intracellular environment for N.
pharaonis includes 4 M KCl, we also investigated the effect of monovalent
and bivalent cationic solutions on pHtrII-cyt CD spectra. In solutions of up to
2 M of either LiCl, NaCl, KCl, RbCl, or CsCl in PBS and CaCl2 and
MgCl2 in buffer B, pHtrII-cyt CD spectra exhibited predominantly unfolded
features. However, at 4 M salt concentrations, helix conformation was
stabilized to different degrees depending on the nature of the salt added (Figure 2(a)). A plot of hydrated ion size (ion radii taken from [35]) versus ellipticity (Figure 2(d)) shows a minimum at the position of
NaCl, indicating that NaCl is best in stabilizing pHtrII-cyt structure. We note that maximum helix induction by the
high salt additions required buffer D (containing phosphate and other salts) as
the base buffer, suggesting that there is additional complexity in the factors
contributing to maximum stability than can be explained by a single solvent
component. This also reflects the complex solvent environment of the halophilic
cytoplasm. For example, studies in H.
marismortui indicated that although KCl was the predominant salt, there
were also considerable amounts of sodium and magnesium salts present [36].Figure 2
(a) Effect of
addition mono- and bivalent salts on far-UV CD spectra of pHtrII-cyt recorded
at protein concentration of 0.1 mg/mL at +20C°. Dotted line is the spectrum of
pHtrII-cyt in PBS, black spectrum corresponds to the addition of 4 M LiCl, red—4 M NaCl, green—4 M KCl, blue—4 M RbCl, cyan—4 M CsCl, magenta—4 M CaCl2, and yellow—4 M MgCl2. (b) Thermal denaturation
plots of pHtrII-cyt monitored at 222 nm in PBS (∘) and in PBS +
monovalent salts  (□). Colors are the
same as in (a). (c) Thermal denaturation plots of pHtrII-cyt monitored at 222
nm in PBS (∘), PBS + 90%(v/v) glycerol (▪), PBS + 98.5%(v/v) TFE (•), and in PBS + 40% saturation
ammonium sulfate (⧫). The straight lines
represent sigmoidal fits of the experimental data. Dependences of the mean
residue molar ellipticity at 222 nm (d) and midpoints of thermal denaturation
monitored at 222 nm (e) of pHtrII-cyt in PBS + 4 M of the corresponding
chloride salt on the hydration radii of alkali cations used
in the study. The straight line in (e) represents linear fit with correlation
coefficient of 0.9. Note that the spectra in the presence of MgCl2 or CaCl2 were recorded in Tris buffer (buffer B, pH 7.2) instead of
PBS because of the low solubility of these salts in PBS.


### 3.5. pHtrII-cyt Structure is Thermally Labile

CD was used to monitor
thermal stability of pHtrII-cyt. The negative ellipticity at 222 nm as a
function of temperature is shown in Figures2(b) and 2(c). In PBS, CD spectra were
essentially independent of temperature, characteristic of random coil at all
temperatures probed (0C° to +99C°). In the presence of 4 M salt (Figure 2(b)),
helicity was lost cooperatively with rising temperature with a single
transition point, observed at +41.5C° in 4 M LiCl, +37.9C° in 4 M NaCl, +36.3C°
in 4 M KCl, and +32.4C° in 4 M RbCl. The melting temperature was thus linearly
dependent on ionic radius of the cations, with smaller ions stabilizing helix
stronger (Figure 2(e)).Denaturation in 90%(v/v) glycerol proceeded similar to that
observed in the presence of high salt concentrations, that is, cooperatively
with a transition point of +38.3C°. Thermal denaturation of pHtrII-cyt in PBS +
40% saturation ammonium sulfate was fitted by a sigmoidal curve with a
transition point significantly higher than those obtained under any other
condition, at +66 ± 3C°. However, the shape of
the curve was not symmetric indicating the presence of multiple species with
different melting temperatures. In 98.5%(v/v) TFE, a transition point of +36 ± 4.5C° was obtained, but seemed to follow a linear
dependency with temperature, suggesting a gradual melting with little
cooperativity.


### 3.6. NMR
Spectroscopy Reveals High Dynamics of pHtrII-cyt

Resonances
in one-dimensionalH1 spectra of pHtrII-cyt in buffer D (data not
shown) were very sharp and clustered around 8.3 ppm. No resonances were
observed upfield of 0.8 ppm. These spectral features are characteristic of
disordered or globally dynamic proteins [37]. H1-N15-HSQC spectra of uniformly N15-labeled
pHtrII-cyt at +10C° and +20C° in buffer D with and without 4 M KCl are
shown in Figures 3(a), 3(b), respectively. Spectra recorded at +4C°, +10C°,
+25C°, +37C°, and +41C° were qualitatively
similar in chemical shift dispersion but varied greatly with respect to
individual chemical shifts (data not shown). A maximum of 254 of the expected
271 backbone NH resonances were resolved at +4C° in buffer D, while only 216
were resolved at +41C° in buffer D. Only 27 peak positions were independent of
temperature for low salt and 44 peak positions for high salt samples. To quantify the
dynamic properties of HtrII-cyt, we measured N15 relaxation parameters T1,T2, and hetNOE
at two different temperatures (+10C° and +20C°) in the presence and absence of
4 M KCl. The results are listed in Table 1. Values could only be obtained for 141–148
of the 251–258 peaks observed in the four respective HSQC spectra, while
106–110 of the peaks decayed so rapidly that reliable relaxation parameters
could not be determined. This indicates that the backbone resonances of these
residues are most likely rigid or undergo conformational exchange on the microsecond-to-millisecond
time scale. For the 141–148 peaks for which relaxation parameters were
determined in buffer D at +10C° and +20C°,
the T1,T2 and hetNOE values are characteristic of random
coil structure as, for example, observed in unfolded lysozyme in 8 M urea [38]. In contrast, the observed T1,T2, and hetNOE values are far from those observed in a rigid helical
protein. There was very little deviation from the average in all relaxation
parameters at both temperatures suggesting that the subset of resonances for
which relaxation parameters could be measured experiences similarly restricted
motions, on the nanosecond-to-subnanosecond time scale. In the presence of 4 M
KCl, hetNOE values were significantly less negative at both temperatures. T2 average values were overall slightly increased as compared to those observed in
the absence of high salt, probably due to the difference in solvent viscosity.
Importantly, however, the standard deviation in T2 times was much
higher in the presence of salt, reflecting the appearance of resonances with
lower T2 times (higher relaxation rates), indicating restriction in
conformational space similar to what has been observed in hydrophobic clusters
in unfolded
lysozyme [38].Table 1
Mean and
standard deviation ofT1,T2, and hetNOE values measured
at different temperatures in buffer D in the presence and absence of 4 M KCl on
a 600 MHz instrument.
Temperature and solventT1(s)T2(s)hetNOE+10C°-buffer D0.67±0.180.28±0.11−0.22±0.54+10C°-buffer D plus 4 M KCl0.66±0.220.33±0.28−0.08±0.30+20C°-buffer
D0.74±0.340.31±0.15−0.35±0.57+20C°-buffer D plus 4 M KCl0.65±0.240.38±0.43−0.12±0.27(a) 600 MHzH1-N15
HSQC spectrum of 5 mg/mL pHtrII-cyt in buffer D plus 4 M KCl at +10C° (red) and
+20C° (black) recorded in thin high salt tubes. Signals correspond to backbone
and side-chain NH or NH2 groups. There is no Trp in the pHtrII-cyt
sequence, so there are no resonances expected beyond 10 ppm. (b) Same as in (a)
measured in buffer D using the same type of NMR tubes.
(a)(b)These results strongly
suggest that both in the presence and absence of salt, there is significant
restriction in the conformational space being explored by the ensemble of
structures. The NMR results provide detailed information on the number of
resonances whose dynamics are affected by changes in solvent conditions. For
141–148 backbone amide groups out of 271 pHtrII-cyt residues (52–55%),
relaxation rates indicated random-coil behavior under low salt conditions.
These represent the most flexible regions of pHtrII-cyt. In these regions, the
presence of 4 M KCl diminishes the degree of flexibility significantly. In the
remaining 45–48% of
resonances, there is stronger restriction in conformational space that is
present both in low and high salt conditions. These residues experience
significant changes in their environment when comparing low and high salt
conditions because the chemical shift values change and a broader range of
values is found under high salt conditions indicating more highly structured
states.


### 3.7. Disorder
Is Predicted to be a Conserved Feature of the MCP Family

The
experimental evidence presented above uniformly suggests that the pHtrII-cyt
structure is highly dynamic. Since the Tar-cyt and Tsr-cyt structures are also
believed to be dynamic (see Section1), we investigated the propensity for
disorder in all three proteins and in the entire MCP family using two methods: PONDR
[31] and RONN [32, 33] (Figure 4). Y-axis values (probability of disorder) higher than 0.5
generally indicate disordered regions. In all three proteins PONDR predicts
substantial disorder for the cytoplasmic domain, while overall the RONN
probabilities for disorder were more moderate (Figure 4(a)). Using RONN, we
investigated the propensity for disorder in the entire MCP family (Figure 4(d)).
A mean value of 0.5 with a low standard deviation of ~0.09 was obtained.
Tsr-cyt and pHtrII-cyt closely follow the mean RONN disorder values in the MCP
family at the majority of sites, indicating that high flexibility but not
complete lack of structure is conserved in the MCP family. Notably, some regions
at cytoplasmic domain alignment position 100–110, 190–200 and at the extreme
C-terminus (>275) displayed least flexibility in the MCP family (Figure 4(d)).
These correspond to regions of lowest B-factors observed in the Tsr-cyt crystal
structure [13].PONDR (red lines)
and RONN (blue lines) predictions of disorder with transmembrane segments
designated by black bars for (a) pHtrII (Swiss-Prot ID P42259), (b) Tsr
(Swiss-Prot ID P02942), (c) Tar
(Swiss-Prot ID P07017), (d)
disorder propensities for MCP family members estimated using RONN. Mean,
maximum, minimum calculated for each position in the alignment are shown as
thin blue lines. The corresponding values for the pHtrII-cyt and the Tsr-cyt
are shown as thick red and green lines, respectively.


## 3.1. pHtrII-cyt
has Strong Propensity for Helix

pHtrII-cyt is predicted to be
a helical coiled-coil based on homology to the serine receptor (Figure1(a)),
high coiled-coil structure propensity (Figure 1(b), top panel), and high helix
propensity with 63% to 97%α-helix content (Figure 1(b),
bottom panel), depending on the prediction algorithm used (see Section 2.5).
These predictions are not consistent with the CD spectra recorded in the
commonly used buffers, PBS, buffer A and buffer B, which were all
indistinguishable and essentially lacked negative ellipticity at 222 nm
characteristic for helix [20]. The values obtained from deconvolution of the spectra were ∼77% unstructured,
∼7% turn, ∼13%β-sheet,
and ∼3%α-helix.
However, control experiments with helix-inducing solvent 2,2,2-trifluoroethanol
(TFE) showed a gradual rise in negative ellipticity at 222 nm with increasing
amounts of TFE added (Figure 1(d)), experimentally validating the high helix
propensity inferred from the pHtrII-cyt sequence.(a) X-ray crystal
structure model of the Tsr-cyt (PDB ID 1QU7) corresponding to identical
positions of the pHtrII-cyt investigated in this study. (b) Top, prediction of
coiled-coil structure for pHtrII-cyt by the COILS software (Section2.5)
and below, prediction of secondary structure for pHtrII-cyt by the DSC software
(Section 2.5). (c) Comparison of FTIR spectra of 10 mg/mL pHtrII-cyt in
buffer C (solid line) and in dry film (dashed line) at room temperature. (d)–(f) Effects
of increasing (d) TFE, (e) glycerol (v/v),
and (f) ammonium sulfate (in % of saturation) concentrations on far-UV CD
spectra of pHtrII-cyt recorded at protein concentration of 0.1 mg/mL at +20C°
(left panel, colors correspond to TFE, glycerol, or ammonium sulfate
concentration) and deduced secondary structure content (right panel, colors
correspond to the content of secondary structure elements). The secondary
structure content estimations were generated by the CONTIN/LL program from the
CDPro package (Section 2.5); secondary structure elements are designated
as follows: helix—black, sheet—red, turn—green, and random coil—blue.
(a)(b)(c)(d)(e)(f)


## 3.2. Dehydration
Stabilizes Helix Structure

Figure1(c) shows FTIR spectra
in the amide I region for pHtrII-cyt in buffer C (solid line) and in dry film
(dashed line). The position of the maximum of the amide I band in buffer C at
1645cm−1 is characteristic of a disordered conformation. In dry
film, a shift to 1654cm−1 was observed, a value that is
characteristic of α-helical secondary structure [25, 34].


## 3.3. Small
Molecule Osmolytes Stabilize Helix Structure

Addition of glycerol to PBS
at concentrations of 60%(v/v) and
higher greatly stabilized helix content which based on spectral deconvolution
rose from ∼19% in 60%(v/v) glycerol
to ∼59% in 90%(v/v) glycerol. β-sheet and turn contributed,
respectively, ∼5% and ∼12% to the spectrum in 90%(v/v) glycerol (Figure 1(e)). In sucrose, at 90% (w/v) saturation, α-helix
content was ∼37%, where β-sheet and turn contributed,
respectively, ∼10% and ∼21%, to the spectrum (data not shown).


## 3.4. Salts
Affect pHtrII-cyt Differentially

Ammonium sulfate, a common
agent to precipitate, stabilize, or facilitate refolding of proteins, up to 30%
saturation, did not significantly alter the CD spectra of pHtrII-cyt obtained
in PBS alone (Figure1(f)). Between 30% and 60%, pHtrII-cyt adopted approximately
50–56%α-helical conformation, while at above 60% saturation, helical content
was reduced and that of random coil increased. Previous crosslinking and
analytical gel-filtration experiments showed that the structural transition was
accompanied by protein aggregation at high ammonium sulfate concentrations [20]. Since the native
intracellular environment for N.
pharaonis includes 4 M KCl, we also investigated the effect of monovalent
and bivalent cationic solutions on pHtrII-cyt CD spectra. In solutions of up to
2 M of either LiCl, NaCl, KCl, RbCl, or CsCl in PBS and CaCl2 and
MgCl2 in buffer B, pHtrII-cyt CD spectra exhibited predominantly unfolded
features. However, at 4 M salt concentrations, helix conformation was
stabilized to different degrees depending on the nature of the salt added (Figure 2(a)). A plot of hydrated ion size (ion radii taken from [35]) versus ellipticity (Figure 2(d)) shows a minimum at the position of
NaCl, indicating that NaCl is best in stabilizing pHtrII-cyt structure. We note that maximum helix induction by the
high salt additions required buffer D (containing phosphate and other salts) as
the base buffer, suggesting that there is additional complexity in the factors
contributing to maximum stability than can be explained by a single solvent
component. This also reflects the complex solvent environment of the halophilic
cytoplasm. For example, studies in H.
marismortui indicated that although KCl was the predominant salt, there
were also considerable amounts of sodium and magnesium salts present [36].Figure 2
(a) Effect of
addition mono- and bivalent salts on far-UV CD spectra of pHtrII-cyt recorded
at protein concentration of 0.1 mg/mL at +20C°. Dotted line is the spectrum of
pHtrII-cyt in PBS, black spectrum corresponds to the addition of 4 M LiCl, red—4 M NaCl, green—4 M KCl, blue—4 M RbCl, cyan—4 M CsCl, magenta—4 M CaCl2, and yellow—4 M MgCl2. (b) Thermal denaturation
plots of pHtrII-cyt monitored at 222 nm in PBS (∘) and in PBS +
monovalent salts  (□). Colors are the
same as in (a). (c) Thermal denaturation plots of pHtrII-cyt monitored at 222
nm in PBS (∘), PBS + 90%(v/v) glycerol (▪), PBS + 98.5%(v/v) TFE (•), and in PBS + 40% saturation
ammonium sulfate (⧫). The straight lines
represent sigmoidal fits of the experimental data. Dependences of the mean
residue molar ellipticity at 222 nm (d) and midpoints of thermal denaturation
monitored at 222 nm (e) of pHtrII-cyt in PBS + 4 M of the corresponding
chloride salt on the hydration radii of alkali cations used
in the study. The straight line in (e) represents linear fit with correlation
coefficient of 0.9. Note that the spectra in the presence of MgCl2 or CaCl2 were recorded in Tris buffer (buffer B, pH 7.2) instead of
PBS because of the low solubility of these salts in PBS.


## 3.5. pHtrII-cyt Structure is Thermally Labile

CD was used to monitor
thermal stability of pHtrII-cyt. The negative ellipticity at 222 nm as a
function of temperature is shown in Figures2(b) and 2(c). In PBS, CD spectra were
essentially independent of temperature, characteristic of random coil at all
temperatures probed (0C° to +99C°). In the presence of 4 M salt (Figure 2(b)),
helicity was lost cooperatively with rising temperature with a single
transition point, observed at +41.5C° in 4 M LiCl, +37.9C° in 4 M NaCl, +36.3C°
in 4 M KCl, and +32.4C° in 4 M RbCl. The melting temperature was thus linearly
dependent on ionic radius of the cations, with smaller ions stabilizing helix
stronger (Figure 2(e)).Denaturation in 90%(v/v) glycerol proceeded similar to that
observed in the presence of high salt concentrations, that is, cooperatively
with a transition point of +38.3C°. Thermal denaturation of pHtrII-cyt in PBS +
40% saturation ammonium sulfate was fitted by a sigmoidal curve with a
transition point significantly higher than those obtained under any other
condition, at +66 ± 3C°. However, the shape of
the curve was not symmetric indicating the presence of multiple species with
different melting temperatures. In 98.5%(v/v) TFE, a transition point of +36 ± 4.5C° was obtained, but seemed to follow a linear
dependency with temperature, suggesting a gradual melting with little
cooperativity.


## 3.6. NMR
Spectroscopy Reveals High Dynamics of pHtrII-cyt

Resonances
in one-dimensionalH1 spectra of pHtrII-cyt in buffer D (data not
shown) were very sharp and clustered around 8.3 ppm. No resonances were
observed upfield of 0.8 ppm. These spectral features are characteristic of
disordered or globally dynamic proteins [37]. H1-N15-HSQC spectra of uniformly N15-labeled
pHtrII-cyt at +10C° and +20C° in buffer D with and without 4 M KCl are
shown in Figures 3(a), 3(b), respectively. Spectra recorded at +4C°, +10C°,
+25C°, +37C°, and +41C° were qualitatively
similar in chemical shift dispersion but varied greatly with respect to
individual chemical shifts (data not shown). A maximum of 254 of the expected
271 backbone NH resonances were resolved at +4C° in buffer D, while only 216
were resolved at +41C° in buffer D. Only 27 peak positions were independent of
temperature for low salt and 44 peak positions for high salt samples. To quantify the
dynamic properties of HtrII-cyt, we measured N15 relaxation parameters T1,T2, and hetNOE
at two different temperatures (+10C° and +20C°) in the presence and absence of
4 M KCl. The results are listed in Table 1. Values could only be obtained for 141–148
of the 251–258 peaks observed in the four respective HSQC spectra, while
106–110 of the peaks decayed so rapidly that reliable relaxation parameters
could not be determined. This indicates that the backbone resonances of these
residues are most likely rigid or undergo conformational exchange on the microsecond-to-millisecond
time scale. For the 141–148 peaks for which relaxation parameters were
determined in buffer D at +10C° and +20C°,
the T1,T2 and hetNOE values are characteristic of random
coil structure as, for example, observed in unfolded lysozyme in 8 M urea [38]. In contrast, the observed T1,T2, and hetNOE values are far from those observed in a rigid helical
protein. There was very little deviation from the average in all relaxation
parameters at both temperatures suggesting that the subset of resonances for
which relaxation parameters could be measured experiences similarly restricted
motions, on the nanosecond-to-subnanosecond time scale. In the presence of 4 M
KCl, hetNOE values were significantly less negative at both temperatures. T2 average values were overall slightly increased as compared to those observed in
the absence of high salt, probably due to the difference in solvent viscosity.
Importantly, however, the standard deviation in T2 times was much
higher in the presence of salt, reflecting the appearance of resonances with
lower T2 times (higher relaxation rates), indicating restriction in
conformational space similar to what has been observed in hydrophobic clusters
in unfolded
lysozyme [38].Table 1
Mean and
standard deviation ofT1,T2, and hetNOE values measured
at different temperatures in buffer D in the presence and absence of 4 M KCl on
a 600 MHz instrument.
Temperature and solventT1(s)T2(s)hetNOE+10C°-buffer D0.67±0.180.28±0.11−0.22±0.54+10C°-buffer D plus 4 M KCl0.66±0.220.33±0.28−0.08±0.30+20C°-buffer
D0.74±0.340.31±0.15−0.35±0.57+20C°-buffer D plus 4 M KCl0.65±0.240.38±0.43−0.12±0.27(a) 600 MHzH1-N15
HSQC spectrum of 5 mg/mL pHtrII-cyt in buffer D plus 4 M KCl at +10C° (red) and
+20C° (black) recorded in thin high salt tubes. Signals correspond to backbone
and side-chain NH or NH2 groups. There is no Trp in the pHtrII-cyt
sequence, so there are no resonances expected beyond 10 ppm. (b) Same as in (a)
measured in buffer D using the same type of NMR tubes.
(a)(b)These results strongly
suggest that both in the presence and absence of salt, there is significant
restriction in the conformational space being explored by the ensemble of
structures. The NMR results provide detailed information on the number of
resonances whose dynamics are affected by changes in solvent conditions. For
141–148 backbone amide groups out of 271 pHtrII-cyt residues (52–55%),
relaxation rates indicated random-coil behavior under low salt conditions.
These represent the most flexible regions of pHtrII-cyt. In these regions, the
presence of 4 M KCl diminishes the degree of flexibility significantly. In the
remaining 45–48% of
resonances, there is stronger restriction in conformational space that is
present both in low and high salt conditions. These residues experience
significant changes in their environment when comparing low and high salt
conditions because the chemical shift values change and a broader range of
values is found under high salt conditions indicating more highly structured
states.


## 3.7. Disorder
Is Predicted to be a Conserved Feature of the MCP Family

The
experimental evidence presented above uniformly suggests that the pHtrII-cyt
structure is highly dynamic. Since the Tar-cyt and Tsr-cyt structures are also
believed to be dynamic (see Section1), we investigated the propensity for
disorder in all three proteins and in the entire MCP family using two methods: PONDR
[31] and RONN [32, 33] (Figure 4). Y-axis values (probability of disorder) higher than 0.5
generally indicate disordered regions. In all three proteins PONDR predicts
substantial disorder for the cytoplasmic domain, while overall the RONN
probabilities for disorder were more moderate (Figure 4(a)). Using RONN, we
investigated the propensity for disorder in the entire MCP family (Figure 4(d)).
A mean value of 0.5 with a low standard deviation of ~0.09 was obtained.
Tsr-cyt and pHtrII-cyt closely follow the mean RONN disorder values in the MCP
family at the majority of sites, indicating that high flexibility but not
complete lack of structure is conserved in the MCP family. Notably, some regions
at cytoplasmic domain alignment position 100–110, 190–200 and at the extreme
C-terminus (>275) displayed least flexibility in the MCP family (Figure 4(d)).
These correspond to regions of lowest B-factors observed in the Tsr-cyt crystal
structure [13].PONDR (red lines)
and RONN (blue lines) predictions of disorder with transmembrane segments
designated by black bars for (a) pHtrII (Swiss-Prot ID P42259), (b) Tsr
(Swiss-Prot ID P02942), (c) Tar
(Swiss-Prot ID P07017), (d)
disorder propensities for MCP family members estimated using RONN. Mean,
maximum, minimum calculated for each position in the alignment are shown as
thin blue lines. The corresponding values for the pHtrII-cyt and the Tsr-cyt
are shown as thick red and green lines, respectively.


## 4. Discussion

### 4.1. A Model for pHtrII-cyt
Structure

Previous studies showed that
the shape of pHtrII-cyt is an elongated rod with a radius of gyration of 54Å [20], both as a monomer in low salt and as a dimer in 4 M KCl. These high KCl concentrations are physiological for N. pharaonis [39, 40]. This shape is consistent with the helical coiled-coil dimer of the
Tsr-cyt crystal structure [13].However, this shape is not consistent with CD data lacking evidence
for helix altogether for the monomer, and indicating only 24% helix for the
dimer in 4 M KCl [20]. Here, we provide evidence that despite “random coil” spectroscopic
features, pHtrII-cyt can exist as a highly flexible, loosely packed but folded
helical coiled-coil: (i) sequence-based structure predictions all strongly
support a helical coiled-coil structure for pHtrII-cyt, and addition of TFE, a
well-known helix inducing agent, is able to generate CD spectra that are
characteristic for ~80% helix; (ii) both, uncharged electrolytes and salts
induce negative ellipticity in CD spectra of pHtrII-cyt, but the thermal
midpoint transitions occur at very low temperatures indicating thermal
instability; (iii) the majority of signals in H1, N15-HSQC spectra
are highly temperature-sensitive, with the largest number of visible peaks
observed at low temperatures and best spectral dispersion in high salt at low
temperatures. Because small angle neutron scattering indicates an identical
radius of gyration [20] regardless of the differences in helix content in CD spectra and
spectral changes in the NMR spectra, we propose that stabilization of more
folded conformations by added stabilizing agents in the case of pHtrII-cyt
means a reduction in dynamics leading to a more compact but overall similar
structure.The stabilization of folded
structures of proteins by additives has been extensively studied beginning with
the seminal work by Hofmeister [41]. Careful experiments, thermodynamic analyses, and discussions over the
years ([42–45] and references within), including those for halophilic proteins [46–49], have led to the relatively recent “unifying theory” that both, “hard”
factors, referring to excluded volume effects and “soft” factors, referring to
interactions between additives, water, and proteins/nucleic acids together
contribute to the mechanisms underlying the effects of cosolvents on
protein/nucleic acid structural stability. While it is possible to empirically
estimate the overall contribution of hard versus soft effects [44], the current uncertainty of the nature of unfolded states will preclude
quantitative modeling in the near future [50]. The very high density of acidic amino acids in pHtrII-cyt typical of
halophilic proteins suggests that “soft” effects will be important in the case
of stabilization of the helical coiled-coil in pHtrII-cyt. To illustrate this
point, we generated homology models of each monomer in the dimer from the Tsr-cyt
crystal structure (Figure 5(a)). FastContact analysis [51] of the model reveals that this dimeric structure is not stable because
of electrostatic repulsion between the acidic residues at the dimer interface
(Figure 5(c)), while each helix in the monomer is favorably packed through hydrophobic
interactions (Figure 5(b)). As a result, docking of the two helical monomers
using ClusPro software [52] does not reproduce the homology model, and instead, only results in
physical interaction in an area in which hydrophobic interactions are possible
(Figure 5(d)). Presumably, the high salt concentrations in vivo overcome the electrostatic repulsions. To test this
hypothesis, we conducted systematic comparison of monovalent and bivalent
cations in the presence of a constant anion (chloride). We found that the
extent and stability of the ellipticity signal in CD spectra in the presence of
different cations follows exactly that predicted by Collins’ law of matching
water affinities [53]: the maximum stabilization is with the ion (Na+) that has a
water affinity closest to those of the acidic amino acid residues in
pHtrII-cyt, and not the one with highest charge density (Ca2+), or
smallest ionic radius (Li+). However, even in the presence of 4 M
NaCl, pHtrII-cyt is still a dynamic ensemble of folded coiled-coil structures,
rather than a single stable conformation.(a) Homology model
of pHtrII-cyt based on the structure of Tsr-cyt. Negatively charged amino acids
are shown in red, positively charged amino acids in blue, and hydrophobic amino
acids in green. (b) FastContact analysis of each helix in a coiled-coil monomer
chain. Amino acids contributing to favorable electrostatic interactions are
shown in cyan, those contributing to free energy contacts in orange, and those
contributing to both are shown in purple. (c) FastContact analysis of the dimer
interface. Colors as in (b), (d). Docking of the two monomeric chains using
ClusPro software. Colors from FastContact analysis as in (b), (c).
(a)(b)(c)(d)


### 4.2. Why Is pHtrII-cyt So Dynamic?

The
dynamic properties of pHtrII-cyt reported here for high salt concentrations
physiological forN. pharaonis are in
good agreement with those reported for Tar-cyt under conditions physiological
for E. coli [16]. Furthermore, Tar-cyt and Tsr-cyt were found to be globally dynamic in
solution [18], similar to our observations with pHtrII-cyt. Finally, disorder
predictions indicate conservation of flexible properties in the entire MCP
family. This evolutionary conservation of flexibility suggests a role in
function. Activation of chemotaxis receptors and phototaxis transducers occurs
through very subtle effects such as small helix movements during activation and
only dimers bind and activate CheA [54–56]. The compaction coupled to dimerization of a preformed ensemble of
coiled-coil helical-like structures proposed here could contribute to the
mechanism for activation. In support of this hypothesis, the strength of the
interaction between helices in the chemoreceptor cytoplasmic domain was found
to modulate kinase activity [54, 55, 57].


### 4.3. Implications
for Study of “Intrinsically Unstructured” Proteins

Recently,
it was estimated that as much as 50% of human proteins belong to the
“intrinsically unstructured” or “natively unfolded” class [58]. These estimates are based on predictors of disorder such as PONDR that
are trained in part on sequence data of proteins with CD, FTIR, or NMR spectra
like the ones we measured for pHtrII-cyt as sole evidence for disorder [59]. The findings presented in this paper supporting that pHtrII-cyt is not
a random coil suggest that the terms “intrinsically unstructured” and “natively
unfolded” would more appropriately be termed “highly dynamic structural
ensemble.” The notion of residual structure is analogous to that observed in
chemically denatured proteins, where it has been proposed that a true random
coil may never exist [60]. The case of pHtrII-cyt is particularly intriguing because its
nonspherical shape allows us to clearly distinguish between a true random coil
that may or may not retain residual structure and a near-native but highly
dynamic structural ensemble. This case also reminds us that local, that is,
secondary structure in the classical sense and long-range tertiary structure
defining overall shape are not independent of each other. Understanding the
amplitude of the motions observed in this and other systems would be an
important future goal towards defining this interrelationship in more detail.
Investigating how much motion is needed to lose the characteristic ellipticipty
in CD spectra would be a good starting point [26].


### 4.4. Relation
to In Vivo Structure and Dynamics

It is an unanswered question
to what extent proteins experimentally observed to be dynamic in vitro are as dynamic in vivo [42, 61]. Evidence suggests that crowding inside the cell can be mimicked with
solutes in vitro. For example, FlgM,
an intrinsically disordered protein gains structure in the presence of high
concentrations of solutes in vitro
and inside living E. coli cells [62]. Low stability of intrinsically unstructured or highly dynamic protein
ensembles has been shown to be related to functions such as membrane insertion
or fusion events, protein interactions, and signal transduction [63].


## 4.1. A Model for pHtrII-cyt
Structure

Previous studies showed that
the shape of pHtrII-cyt is an elongated rod with a radius of gyration of 54Å [20], both as a monomer in low salt and as a dimer in 4 M KCl. These high KCl concentrations are physiological for N. pharaonis [39, 40]. This shape is consistent with the helical coiled-coil dimer of the
Tsr-cyt crystal structure [13].However, this shape is not consistent with CD data lacking evidence
for helix altogether for the monomer, and indicating only 24% helix for the
dimer in 4 M KCl [20]. Here, we provide evidence that despite “random coil” spectroscopic
features, pHtrII-cyt can exist as a highly flexible, loosely packed but folded
helical coiled-coil: (i) sequence-based structure predictions all strongly
support a helical coiled-coil structure for pHtrII-cyt, and addition of TFE, a
well-known helix inducing agent, is able to generate CD spectra that are
characteristic for ~80% helix; (ii) both, uncharged electrolytes and salts
induce negative ellipticity in CD spectra of pHtrII-cyt, but the thermal
midpoint transitions occur at very low temperatures indicating thermal
instability; (iii) the majority of signals in H1, N15-HSQC spectra
are highly temperature-sensitive, with the largest number of visible peaks
observed at low temperatures and best spectral dispersion in high salt at low
temperatures. Because small angle neutron scattering indicates an identical
radius of gyration [20] regardless of the differences in helix content in CD spectra and
spectral changes in the NMR spectra, we propose that stabilization of more
folded conformations by added stabilizing agents in the case of pHtrII-cyt
means a reduction in dynamics leading to a more compact but overall similar
structure.The stabilization of folded
structures of proteins by additives has been extensively studied beginning with
the seminal work by Hofmeister [41]. Careful experiments, thermodynamic analyses, and discussions over the
years ([42–45] and references within), including those for halophilic proteins [46–49], have led to the relatively recent “unifying theory” that both, “hard”
factors, referring to excluded volume effects and “soft” factors, referring to
interactions between additives, water, and proteins/nucleic acids together
contribute to the mechanisms underlying the effects of cosolvents on
protein/nucleic acid structural stability. While it is possible to empirically
estimate the overall contribution of hard versus soft effects [44], the current uncertainty of the nature of unfolded states will preclude
quantitative modeling in the near future [50]. The very high density of acidic amino acids in pHtrII-cyt typical of
halophilic proteins suggests that “soft” effects will be important in the case
of stabilization of the helical coiled-coil in pHtrII-cyt. To illustrate this
point, we generated homology models of each monomer in the dimer from the Tsr-cyt
crystal structure (Figure 5(a)). FastContact analysis [51] of the model reveals that this dimeric structure is not stable because
of electrostatic repulsion between the acidic residues at the dimer interface
(Figure 5(c)), while each helix in the monomer is favorably packed through hydrophobic
interactions (Figure 5(b)). As a result, docking of the two helical monomers
using ClusPro software [52] does not reproduce the homology model, and instead, only results in
physical interaction in an area in which hydrophobic interactions are possible
(Figure 5(d)). Presumably, the high salt concentrations in vivo overcome the electrostatic repulsions. To test this
hypothesis, we conducted systematic comparison of monovalent and bivalent
cations in the presence of a constant anion (chloride). We found that the
extent and stability of the ellipticity signal in CD spectra in the presence of
different cations follows exactly that predicted by Collins’ law of matching
water affinities [53]: the maximum stabilization is with the ion (Na+) that has a
water affinity closest to those of the acidic amino acid residues in
pHtrII-cyt, and not the one with highest charge density (Ca2+), or
smallest ionic radius (Li+). However, even in the presence of 4 M
NaCl, pHtrII-cyt is still a dynamic ensemble of folded coiled-coil structures,
rather than a single stable conformation.(a) Homology model
of pHtrII-cyt based on the structure of Tsr-cyt. Negatively charged amino acids
are shown in red, positively charged amino acids in blue, and hydrophobic amino
acids in green. (b) FastContact analysis of each helix in a coiled-coil monomer
chain. Amino acids contributing to favorable electrostatic interactions are
shown in cyan, those contributing to free energy contacts in orange, and those
contributing to both are shown in purple. (c) FastContact analysis of the dimer
interface. Colors as in (b), (d). Docking of the two monomeric chains using
ClusPro software. Colors from FastContact analysis as in (b), (c).
(a)(b)(c)(d)


## 4.2. Why Is pHtrII-cyt So Dynamic?

The
dynamic properties of pHtrII-cyt reported here for high salt concentrations
physiological forN. pharaonis are in
good agreement with those reported for Tar-cyt under conditions physiological
for E. coli [16]. Furthermore, Tar-cyt and Tsr-cyt were found to be globally dynamic in
solution [18], similar to our observations with pHtrII-cyt. Finally, disorder
predictions indicate conservation of flexible properties in the entire MCP
family. This evolutionary conservation of flexibility suggests a role in
function. Activation of chemotaxis receptors and phototaxis transducers occurs
through very subtle effects such as small helix movements during activation and
only dimers bind and activate CheA [54–56]. The compaction coupled to dimerization of a preformed ensemble of
coiled-coil helical-like structures proposed here could contribute to the
mechanism for activation. In support of this hypothesis, the strength of the
interaction between helices in the chemoreceptor cytoplasmic domain was found
to modulate kinase activity [54, 55, 57].


## 4.3. Implications
for Study of “Intrinsically Unstructured” Proteins

Recently,
it was estimated that as much as 50% of human proteins belong to the
“intrinsically unstructured” or “natively unfolded” class [58]. These estimates are based on predictors of disorder such as PONDR that
are trained in part on sequence data of proteins with CD, FTIR, or NMR spectra
like the ones we measured for pHtrII-cyt as sole evidence for disorder [59]. The findings presented in this paper supporting that pHtrII-cyt is not
a random coil suggest that the terms “intrinsically unstructured” and “natively
unfolded” would more appropriately be termed “highly dynamic structural
ensemble.” The notion of residual structure is analogous to that observed in
chemically denatured proteins, where it has been proposed that a true random
coil may never exist [60]. The case of pHtrII-cyt is particularly intriguing because its
nonspherical shape allows us to clearly distinguish between a true random coil
that may or may not retain residual structure and a near-native but highly
dynamic structural ensemble. This case also reminds us that local, that is,
secondary structure in the classical sense and long-range tertiary structure
defining overall shape are not independent of each other. Understanding the
amplitude of the motions observed in this and other systems would be an
important future goal towards defining this interrelationship in more detail.
Investigating how much motion is needed to lose the characteristic ellipticipty
in CD spectra would be a good starting point [26].


## 4.4. Relation
to In Vivo Structure and Dynamics

It is an unanswered question
to what extent proteins experimentally observed to be dynamic in vitro are as dynamic in vivo [42, 61]. Evidence suggests that crowding inside the cell can be mimicked with
solutes in vitro. For example, FlgM,
an intrinsically disordered protein gains structure in the presence of high
concentrations of solutes in vitro
and inside living E. coli cells [62]. Low stability of intrinsically unstructured or highly dynamic protein
ensembles has been shown to be related to functions such as membrane insertion
or fusion events, protein interactions, and signal transduction [63].



---

*Source: 267912-2008-10-16.xml*