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Originally published In Press as doi:10.1074/jbc.M505435200 on September 14, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38556-38561, November 18, 2005
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Phenylalanine Side Chain Behavior of the Intestinal Fatty Acid-binding Protein

THE EFFECT OF UREA ON BACKBONE AND SIDE CHAIN STABILITY*{boxs}

Hua Li and Carl Frieden1

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, May 18, 2005 , and in revised form, August 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The equilibrium unfolding behavior of the intestinal fatty acid-binding protein has been investigated by 19F-NMR after incorporation of 4-fluorophenylalanine and by pulsed field gradient diffusion 1H-NMR. At low urea concentrations (0-3 M) but prior to the global unfolding that begins at 4 M urea, the protein exhibits dynamic motion in the backbone and an expanded hydrodynamic radius with no major change in the side chain orientation. As monitored by two-dimensional 19F-19F nuclear Overhauser effect, the distance between two phenylalanine residues (Phe68 and Phe93) located in the two different {beta}-sheets that enclose the internal cavity did not change up to 4 M urea. Additionally, the chemical shifts of these two residues changed almost identically as a function of denaturant. At all urea concentrations, as well as in the native protein, multiple conformations exist. These conformers interconvert at different rates under different conditions, ranging from slow exchange by showing separate peaks in the native state to intermediate exchange at intermediate urea concentrations. Residual structure persisted around Phe62 even at very high concentrations of denaturant, suggesting that region as a nucleation site during folding. The results were compared with previous studies examining the backbone behavior (Hodsdon, M. E., and Frieden, C. (2001) Biochemistry 40, 732-742) and suggest that the side chains show more stability than the backbone prior to global unfolding of the protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Incorporating fluorine-labeled amino acids into proteins has allowed investigation of the effects of ligand addition, conformational changes, and protein folding on the behavior of side chains (1, 2). Such data should be complementary to data examining the response of the backbone to these effects by methods such as, for example, circular dichroism or hydrogen/deuterium (H/D)2 exchange. Previous studies from our laboratory using 19F-labeled amino acids have focused on the stabilization of side chains during the folding process and have found that, in general, such stabilization appears to be associated with the last step in the formation of the native structure (3, 4). Examination of side chain behavior of fluorine-labeled residues, however, can also be used to monitor the behavior and relative distances between the side chains of labeled residues under various conditions.

The intestinal fatty acid-binding protein (IFABP) is one of a class of small (15 kDa) proteins that bind ligands into a large cavity surrounded by 10 antiparallel {beta}-strands. IFABP contains eight phenylalanine residues, and all of them are included in the 29 residues that line this binding cavity (5) (Fig. 1). We have previously assigned the 19F-NMR resonances for each residue (6). Monitoring the behavior of each phenylalanine under a variety of conditions should allow the ability to compare and contrast the role of the different phenylalanine residues.

In the present work we have examined the role of phenylalanines in protein stability by measuring their 19F-NMR properties as a function of urea concentration. Combined with the global property from PFG diffusion experiments, the results are compared with 1H-15N heteronuclear single quantum coherence (HSQC) and H/D exchange data obtained earlier as a function of urea concentration (7). Here we show that side chain behavior may differ from backbone behavior with respect to unfolding by urea under equilibrium conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Ultrapure urea was purchased from United States Biochemical. The concentration of urea was determined by index of refraction at 25 °C (8). 4-19F-Phe was obtained from Acros Organics (Morris Plains, NJ). All other chemicals were of reagent grade.

Protein Production and Purification—The unlabeled and 4-19F-labeled IFABP was produced and purified as described elsewhere (6). Protein in the NMR experiments was used within ~2 weeks of its preparation.

Sample Preparation for 19F-NMR—For one-dimensional NMR spectra as a function of urea, the samples were made by dilution of 2.4 mM stock protein solution containing 20 mM potassium phosphate and 0.25 mM EDTA, pH 7.3 (NMR buffer) and the same buffer in 10.2 M urea to give a final concentration of 200 µM protein at different urea concentrations. For two-dimensional spectra, the samples were made similar to those for one-dimensional spectra with protein concentration of 1 mM unless otherwise indicated. All of the samples were made by adding the protein stock to the premixed solution.

Sample Preparation for PFG Diffusion 1H-NMR—The stock protein solution in H2O was lyophilized and redissolved in D2O and then lyophilized three more times from D2O. The NMR buffer and urea stock buffer were treated the same way. The protein was finally dissolved in 99.99% D2O. The samples in urea were made by diluting stock protein solution in D2O into the NMR buffer and 10.5 M urea in D2O to give a final concentration of 1 mM at different urea concentrations. To each sample, 2.5 µl of 100 mM dioxane solution in D2O was added to act as an internal viscosity standard. Shigemi NMR tubes, with 1.5-cm sample height, were used to ensure the linearity of gradient.



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  		FIGURE 1.
The crystal structure (Protein Data Bank entry 1IFB [PDB] ) of apo-IFABP showing the location of eight phenylalanine residues. The diagram was prepared using MolMol (25).

 
19F-NMR Spectroscopy—19F-NMR spectra were acquired on a Varian Unity-Plus 500 MHz spectrometer operating at 470.3 MHz with a Varian Cryo-Q dedicated to a 5-mm 19F probe without 1H decoupling. The 19F probe was cooled and kept at 20 K with the Varian Cryo-Q open cycle cryogenic system. All one-dimensional spectra were recorded at 20 °C with 64 scans and processed by NMRPipe (9) with 12-Hz exponential line broadening unless otherwise indicated. For quantitative measurements (relative to the intensity of 6-19F-tryptophan) a delay of 5 s was used. Under these conditions, no saturation of 19F signals was observed. For the two-dimensional NOESY experiments at different urea concentrations, the mixing time was selected by acquiring a series of NOESY spectra with different mixing times from 50 to 500 ms. An optimal value of 250 ms was used to probe the distance change between Phe68 and Phe93 as a function of urea concentration. The two-dimensional NMR data were analyzed by the PIPP program (10). The spectra were referenced to an internal standard of 6-19F-tryptophan (~0.2 mM; the actual chemical shift is -46.293 ppm relative to trifluoroacetic acid), and all samples contained 8% (v/v) D2O. There was no correction to the pH value due to D2O.

PFG Diffusion 1H-NMR Spectroscopy—PFG-NMR diffusion measurements were made with a bipolar PFG longitudinal eddy current delay pulse sequence (11) on a Varian Inova 500 spectrometer at 20 °C. Twenty-two spectra were acquired with the strength of the diffusion gradient varying between 2.5 and 100% of its maximum value. The length of the diffusion gradient was optimized for each sample to give a total decay of ~90% of the initial intensity. For protein samples at 0, 1.5, 2.5, and 4 M urea the length of the diffusion gradient was set to 3 ms, whereas in 5.5 and 7 M urea it was set to 5 ms. The echo time for diffusion was set to 150 ms, and all spectra were acquired with 10,000 complex points and a sweep width of 7000 Hz. Because many contaminants in protein solution give rise to resonances in the aliphatic region of the spectrum, only the integration in the aromatic region (6.5-8.2 ppm) was used as a function of gradient strength, following standard methods (12). The reported hydrodynamic radius (Rh) of dioxane, 2.12 Å (13), was used to calculate the Rh of IFABP under different urea concentrations.

Fluorescence Spectra—Steady state fluorescence experiments as a function of urea were carried out at 20 °C using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, NJ). The excitation wavelength used was 290 nm, and the emission spectra were recorded between 300 and 400 nm. Equilibrium data were fit to a two-state model as described by Ropson et al. (14) with an equation from Santoro and Bolen (15).



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  		FIGURE 2.
Equilibrium unfolding of 19F-Phe-IFABP as a function of urea concentration as monitored by fluorescence. The experiment was performed with 2 µM protein in 20 mM potassium phosphate buffer and 0.25 mM EDTA at pH 7.3 and 20 °C. The excitation and emission wavelengths were set to 290 and 327 nm, respectively. The solid line is a fit to the data as described previously (15). The midpoint of the denaturation curve is 4.9 M.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Denaturation of Labeled IFABP—Fig. 2 shows the change in intrinsic fluorescence of fully labeled IFABP with urea concentration. The data were obtained under similar conditions as used in NMR experiments except at much lower protein concentrations. Fully labeled 19F-Phe-IFABP has a denaturation midpoint (4.9 M) that is slightly higher than that obtained for wild-type protein (~4.8 M) (16). We have shown that circular dichroism data are superimposable on the fluorescence data (not shown), indicating that the fluorescence data monitor global unfolding. For the discussion below, it is important to note that the global unfolding of the protein only begins at >4 M urea.

One-dimensional 19F-NMR Spectra of IFABP as a Function of Urea Concentration—The equilibrium unfolding of fully labeled IFABP was studied by 19F-NMR as a function of urea concentration. Fig. 3A shows the spectra acquired at different urea concentrations for fully labeled IFABP. The assignments for each of the phenylalanine residues has been made previously (6). At urea concentrations >3 M the decrease in native peaks is accompanied by an increase in unfolded peaks around -40.5 ppm, characteristic of the denatured resonances and indicating that the exchange between native form and denatured forms is slow on the NMR time scale. Fig. 3B shows the chemical shift changes as a function of urea concentration. It is obvious that major chemical shift changes occur at <3 M urea, well before the protein begins to undergo global unfolding. All phenylalanines show a similar denaturation midpoint of ~4 M urea (data not shown). The midpoint is well above the urea concentrations influencing the chemical shifts and well below that for the global unfolding.

Other changes occur at higher urea concentrations. Careful examination of Fig. 3A shows, for example, that a new small peak for Phe62,in addition to the major peak at -47.15 ppm, appeared at -46.72 ppm in 0.5 M urea, indicating the presence of a new conformation. The intensity of these two peaks (at -46.72 and -47.15 ppm) initially decreases with urea but increases at urea concentrations of >5 M, concentrations at which all other native peaks are gone (supplemental Fig. 1, available in the on-line version of this article). Although not obvious from Fig. 3A (because the intensities of the spectra from 5 M to 8.5 M urea have been reduced in the figure for plotting purposes), there is a loss of the total intensity between 3.5 and 6 M urea, which is discussed below.



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  		FIGURE 3.
Equilibrium unfolding data. A, 19F-NMR spectra of apo-IFABP as a function of urea. Each spectrum was recorded at 20 °C with 64 scans and processed with 12 Hz exponential line broadening. Data were obtained at 200 µM protein in 20 mM potassium phosphate buffer (pH 7.3 containing 0.25 mM EDTA) with various concentrations of urea. The peak (marked with an asterisk) at -46.293 ppm is 6-19F-tryptophan used as a reference. Note that the spectra from 5 M-8.5 M urea have been reduced for plotting purposes. B, chemical shift changes of 19F-Phe-IFABP as a function of urea concentration.

 
In Fig. 3A, Phe68 and Phe93 behave differently by exhibiting broader line widths than those for the other residues. Our previous study has shown that Phe68 has two conformations at native state, as it could be deconvoluted into two peaks. The same is true for Phe93 at temperatures of >34 °C (6). To further characterize the urea denaturation behavior, we used proteins singly labeled either at Phe68 or Phe93 (Fig. 4), as well as the protein labeled at both Phe68 and Phe93 (supplemental Fig. 2, available in the on-line version of this article). These proteins show a slightly lower denaturation midpoint than does the fully labeled protein. For protein singly labeled at Phe68, the separation of the two resonances, corresponding to the two conformations, is slightly more extensive than that for the fully labeled protein. Also, the major upfield conformation is less stable because it decreases more rapidly with increasing urea (Fig. 4A). Multiple resonances were also observed for Phe93 and Phe68 at higher urea concentrations around the denatured region, -40.5 ppm (Fig. 4, B and C, respectively), with one peak continuing to increase whereas the others decrease until merging with noise. Those decreasing peaks represent some unfolded like intermediates in slow exchange with the unfolded conformers.



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  		FIGURE 4.
Urea effect on the spectrum of singly labeled IFABP at positions Phe68 or Phe93. A, the change of native (or native-like) resonances of Phe68 with urea. The scale for each spectrum is from -44.2 ppm to -47.3 ppm. B and C, the change of denatured resonances of Phe93 and Phe68 with urea, respectively. The scale for each spectrum in panels B and C is from -39.3 to -41.3 ppm.

 
Homonuclear 19F-Nuclear Overhauser Effect between 4-19F-Phe68 and 4-19F-Phe93 as a Function of Urea Concentration—From the crystal structure (17) one can estimate that the distance between the 19F nuclei of Phe68 and Phe93 is ~2 Å. The relative distance change with urea can be determined by the relationship I {alpha} 1/r6 (18), where I is the intensity of the cross peak and r is the distance between two nuclei that gives rise to the nuclear Overhauser effect cross-peak. Using two-dimensional NOESY measurements, it is possible to monitor the distance between 4-19F-Phe68 and 4-19F-Phe93 at 0, 1.5, 2.5, and 4 M urea (Fig. 5). A crosspeak between Phe68 and Phe93 appears at all these urea concentrations. The peak intensities at 1.5, 2.5, and 4 M urea were normalized relative to that at 0 M urea by dividing the intensities by the percentage of native structure. The latter was normalized relative to that at 0 M urea by integrating five different regions of the spectra, namely, the combined integration of Phe2, Phe17, and Phe93, the integration of Phe47, Phe62, and Phe68 separately, and the integration between -40.3 and -41.3 ppm (Fig. 3A), and all gave similar results. The average was used as the percentage of native structure. From 0 to 4 M urea, the distance between 4-19F-Phe68 and 4-19F-Phe93 barely changes (supplemental TABLE ONE, available in the on-line version of this article). Thus, any native (or native-like) structures that remain show little change in the distance between the two residues.



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  		FIGURE 5.
Two-dimensional NOESY spectra of 4-19F-Phe labeled IFABP at different urea concentrations. The spectra were recorded at 20 °C with 1 mM protein in 20 mM potassium phosphate buffer (pH 7.3, containing 0.25 mM EDTA) containing different concentrations of urea. The mixing time was set to 250 ms and 100 points were used in the F1 dimension.

 
Under native conditions, minor conformations, with ~0.1-0.5% of the population for Phe47 and Phe62 and ~8% of the population for Phe2 were observed by the appearance of exchange cross-peaks in phasesensitive 19F-NOESY (6) (Fig. 5). With increasing urea the exchange cross-peaks for these two conformations get weaker for two reasons. First, the overall concentration of native-like structure is decreased with the increasing urea and, second, the population of the minor conformation is very low and the resonance of minor conformation broadens relative to the major form (19).

PFG Diffusion 1H-NMR as a Function of Urea—The apparent hydrodynamic radius of IFABP increases substantially at high concentrations of urea (TABLE ONE), indicating protein unfolding. The hydrodynamic radii at 1.5 and 2.5 M urea increased ~ 10%, which is an ~33% increase in hydrodynamic volume even though major chemical shift changes occur for most resonances (Fig. 3B) without global unfolding (Fig. 2).


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  		TABLE ONE
Hydrodynamic radii for 1 mM IFABP at different concentrations of urea

 
As intermediates undetectable by 19F-NMR are expected to be beyond detection by diffusion NMR, the intensity loss observed by PFG-NMR comes from two factors, namely increase of gradient and exchange broadening. Therefore, we expect the midpoint determined by PFG-NMR to be similar to that determined by 19F-NMR. Because the intermediates were not detectable we can assume that only two states exist during unfolding, and we can use the experimentally determined decay rates for the native state (dN) and unfolded state (dU) to fit the NMR intensity as a function of gradient strength under different urea concentrations using Equation 1,

(Eq. 1)
where AN and AU are the relative population of the native and denatured states, respectively. At 4 M urea, this analysis gives values of 51.5 ± 2.8% for AN and 50.8 ± 1.2% for AU. The unfolding midpoints obtained by 19F-NMR and PFG-NMR are the same.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Structural Changes Prior to Global Unfolding—It is instructive to compare the data dealing with side chain behavior obtained in this study with data from previous NMR studies of IFABP dealing with backbone behavior as a function of urea concentration. As an example, data with respect to Phe68 are shown in Fig. 6. Data for the other phenylalanine residues (not shown) are essentially similar to those shown in the Fig. 6. Fundamentally, what this comparison shows is that the backbone, at least for phenylalanine residues, behaves differently from the side chains.

Hodsdon and Frieden (7) monitored amide H/D exchange rate and collected a series of two-dimensional 1H-15N-HSQC spectra to examine the properties of the backbone as a function of urea concentration (7). That study made several points. First, all amide H/D exchange rates increased significantly at low urea. Several of the backbone amide protons have exchange times on the order of days, but it was found that by slightly over ~2 M urea all amides exchanged too rapidly to be measured within experimental dead time (about 10 m). This result is shown by the leftmost curve (-x-) in Fig. 6. The data suggest that, at low urea concentrations, amide protons become more susceptible to exchange as a consequence of the protein undergoing a conformational change from a closed form (e.g. H-bonded) to an open form (20). Second, except for regions of residual structure, all of the native backbone resonances show midpoints between 2 and 3 M, disappearing by ~4 M urea. These data indicate that amide resonances are broadened by conformational fluctuations on a millisecond to microsecond time scale. The HSQC data for Phe68 are shown in Fig. 6 and can be interpreted to reflect a transition from a well formed secondary structure to one or more intermediate forms with a more dynamic backbone. It is important to note that these changes occur without any appreciable fluorescence (or circular dichroism) change.

At higher urea concentrations, but prior to global unfolding, the resonances for the 19F-labeled phenylalanines start to disappear. These data suggest that the stability of the hydrophobic phenylalanine side chains persists even after the backbone becomes more dynamic. Finally, as measured by fluorescence, the protein undergoes global unfolding.

It is intriguing to examine the behavior of the protein between the urea concentration where the H/D exchange is rapid and the concentration where global unfolding starts. This is the region where major chemical shift changes of phenylalanines occur (2.5-3.0 M urea), H/D exchange is fast, most backbone amide resonances are broadened by at least half, and the hydrodynamic radius is increased by ~10%. These phenomena suggest that the native structure (or native-like) has become more expanded and dynamic. These expanded conformations could be very similar to the mechanically softened conformations under low denaturant concentrations observed by atomic force microscopy (21). The chemical shift changes also indicate some structural perturbation of the expanded conformations.



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  		FIGURE 6.
Normalized data for Phe68 with respect to hydrogen/deuterium exchange (-X-), loss of amplitude of HSQC signal (-{diamond}-), loss of amplitude of 19F-Phe68 (-{square}-) and loss of overall fluorescence intensity (-{circ}-). The hydrogen/deuterium exchange data reflect the half-times of exchange. By slightly over 2 M urea the rate was too fast to measure by the techniques used (7). These data and the HSQC data are taken from Hodsdon and Frieden (7).

 
Although no good theory exists to correlate fluorine chemical shift with environment, a systematic chemical shift change with the change of conditions may give some clues provided that the structure is known. As a function of urea the 19F chemical shifts of Phe68 and Phe93, whose distance remains at ~ 2Åupto4 M urea, change downfield in an almost identical way. This observation may indicate that they experience the same or very similar environmental changes. Because these two strands represent two different {beta}-sheets, the overall structure remains up to the point of global unfolding. The behavior of Phe47 and Phe62 is somewhat different from that of Phe68 and Phe93, showing much smaller chemical shift changes with urea and suggesting that these regions may experience fewer environmental changes at low urea concentrations. As with Phe68 and Phe93, Phe62 and Phe47 are spatially close.

All of the phenylalanine residues, except Phe55, are located in structural elements other than turns. In particular, Phe47, Phe62, Phe68 and Phe93 are stacked around one of the critical turns between the D and E strands. The observed changes relate to the mechanism of protein unfolding showing that there are no major changes in side chain orientation prior to global unfolding but rather movement of structural regions of the protein.

Evidence of Intermediates during Urea Unfolding—The denaturation midpoint observed by 19F-NMR is lower than that observed by fluorescence. We should emphasize, however, that the discrepancy does not necessarily mean that phenylalanines globally destabilize before tryptophans do. The difference could be a consequence of the fact that any folding intermediate that is exchange-broadened would be undetectable on the NMR time scale and therefore would be recorded as an unfolded state, yielding an apparent lower midpoint compared with global unfolding. In the case of IFABP, there is no evidence of residual structure in the unfolded form around tryptophan measured by fluorescence (22), but there is strong evidence that intermediates exist during urea unfolding by 19F-NMR. In Fig. 3A there is significant intensity loss (~20 to ~30%) from ~3.5 to 6 M urea (compared with the reference peak of 6-19F-Trp at -46.293 ppm). However, no substantial change in the line width was observed for the native peaks or denatured peaks during the urea titration course, indicating the intensity loss was not caused by exchange between unfolded states and folded states. The most reasonable explanation for the intensity loss is the presence of intermediates exchanging on a time scale undetectable by NMR, with those intermediates being more like the unfolded state. In Fig. 3A (and supplemental Fig. 2), the sharpening and the increasing intensity of the resonances of the unfolded states also points to the existence of NMR-undetectable intermediates.

Residual Structures at High Denaturant Concentrations—The central issue in the mechanism of protein folding is the nature of the nuclei around which the protein folds. The common perception is that such nuclei are more stable and may show persistent structure under unfolding conditions. In previous work, we have identified several regions for nuclei formation based on single site mutations (23, 24) as well as NMR studies (7, 22). In general the data have been consistent in proposing turns between {beta}-strands as nucleation spots.

As shown in Fig. 3A (see also supplemental Fig. 1), two native-like peaks (at -46.72 ppm and -47.15 ppm) persist above urea concentration of 5 M urea, concentrations at which all other native peaks are essentially gone. These data suggest that a core region remains collapsed in the most hydrophobic region even when the majority of the protein is completely unfolded. The data also indicate that Phe62 and other hydrophobic residues are involved in hydrophobic collapse during the very early stage of folding. This result is consistent with other studies indicating residual structure near this region (7, 22).

The chemical shift of Phe62 is the most upfield, possibly due to its being surrounded by other aromatic side chains. At higher urea concentrations (e.g. 8.5 M) residual Phe62 still resonates upfield the most (-46.72 ppm), only slightly differently than in its native state (-47.15 ppm). This large shielding effect may also indicate that the side chain topology around Phe62 is largely maintained at higher urea concentration.

Conclusions—Altogether, the current data in combination with previous results suggest the following structural changes as a function of urea concentrations. At low urea, chemical shift changes and H/D exchange experiments indicate that the structure is loosened to an extent to allow solvent penetration. As the urea concentration increases, the backbone structure may convert from rigid, well formed structures to intermediate states that are poorly formed or more dynamic. Little or no fluorescence change occurs when the backbone undergoes these transitions. The phenylalanine side chains remain organized until just prior to global unfolding, suggesting that clusters of hydrophobic residues remain stable until global unfolding occurs. Substantial intermediates are present between 3.5 M and 6 M urea. Even after the protein unfolds, some transient structural regions exist at high denaturant concentrations.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK13332. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental TABLE ONE (relative distance change between 4-19F-Phe68 and 4-19F-Phe93 as a function of urea concentration), supplemental Fig. 1 (intensity and chemical shift change of Phe62 as a function of urea concentration), and supplemental Fig. 2 (19F-NMR spectra of apo-IFABP with 19F-label on both Phe68 and Phe93 as a function of urea concentration. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63110. Tel.: 314-362-3344; Fax: 314-362-7183; E-mail: frieden{at}biochem.wustl.edu.

2 The abbreviations used are: H/D, hydrogen/deuterium; HSQC, heteronuclear single quantum coherence; IFABP, intestinal fatty acid binding protein; NOESY, nuclear Overhauser effect spectroscopy; PFG, pulsed field gradient. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Andre D'Avignon for discussions about pulse field gradient experiments and Robert Horton for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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