effect of guanidine hydrochloride on the hydrodynamic and ... · that of the solvent, respectively....

Effect of Guanidine Hydrochloride on the Hydrodynamic and Thermodynamic Properties of Human Apolipoprotein A-I in Solution*

(Received for publication, November 5, 1979, and in revised form, February 14, 1980)

Celina Edelstein and Angelo M. Scanu From the Departments of Medicine and Biochemistry, The University of Chicago, Pritzker School of Medicine, Chicago, Illinois 60637

To further define its properties in solution, the course of denaturation of apolipoprotein A-I (apo-A-I) was studied as a function of various concentrations of guanidine hydrochloride (GdmCl) using the techniques of circular dichroism, ultraviolet difference spectroscopy, analytical ultracentrifugation, viscometry, and densi- tometry. At low molarities of GdmCl (0 to 0.4 M), apo- A-I exhibited anomalous ellipticity changes which were dependent upon apo-A-I concentration; from ultracentrifugal studies, these changes were interpreted as related to the dissociation of protein oligomers into a new equilibrium attended by a preferential interaction with GdmCl (0.07 g/g of apo-A-I and an increase in molar volume, 197 ml). On the other hand, the frictional (1.68) and axial (5.7) ratios were similar to those in the native state. At molarities of GdmCl between 0.4 M and 6 M, apo-A-I dissociated into monomers by a cooperative denaturation process which was independent of protein concentration: both dissociation and denaturation were thermodynamically reversible with an apparent free energy of stabilization, = 3.70 kcal/mol. Between 2 and 6 M GdmC1, changes in the conformation, intrinsic viscosity, and volume of apo-A-I were noted as assessed by spectroscopic, viscometric, and partial specific volume measurements. These changes ap- peared related to the number of moles of GdmCl bound to apo-A-I (at 2 M GdmC1, 63 mol; at 6 M, 181 mol).

The results indicate that the denaturation of apo-A-I by GdmCl is reversible but follows a complex course occurring in at least two stages; the initial one involv- ing the dissociation of oligomers into monomers and the second one the unfolding of these monomers associated with an increase of their binding to GdmC1.

The study of the physical and chemical properties of apolipoprotein A-I, the major protein constituent of high density lipoprotein, has been the subject of numerous investigations in various laboratories (1, 2). The process of self-association for this protein has been examined (3-5), and the formation of oligomers documented at protein concentrations above 0.2 g/ liter. It has also been observed that at this concentration self- association influences the extent and nature of the interactions between apo-A-I’ and lipids (6). Recently, the stability of apo- A-I in aqueous solutions has been investigated (7-10). By

* This work was supported by United States Public Health Serivce Program Project Grant HL 18577. 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 accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

I The abbreviations used are: apo-A-I, apolipoprotein A-I; GdmC1, guanidine hydrochloride.

exposing apo-A-I to denaturing conditions, Reynolds (9) and Tall et al. (10) concluded that apo-A-I is loosely folded in solution; moreover, calorimetric studies by Tall et al. (10) provided further evidence for a low free energy of stabilization (AG = 2.7 kcal/mol) for this protein.

Stimulated by these observations and in an attempt to better define the molecular events attending the denaturation of apo-A-I in solution, we embarked on a detailed analysis of the structural stability of human apo-A-I as a function of its concentration in solution as well as a function of guanidine hydrochloride. In particular, our attention was directed at defining the structural changes that occur when this apoprotein is titrated against various molar concentrations of GdmCl and at determining the hydrodynamic and thermodynamic parameters of the denaturation process by utilizing spectroscopic and ultracentrifugal methods as well as viscometric and partial specific volume measurements. An account on these studies is the subject of this report.

MATERIALS AND METHODS

Purification of Apo-A-I

Apo-A-I was obtained from delipidated human serum high density lipoprotein’ by molecular sieve chromatography in 8 M urea as previously described (11). Purity was ascertained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea, by the lack of precipitation reaction with anti-apo-A-I1 and anti- apo-C peptides, and by amino acid analysis.

Denaturation Studies

Studies with GdmCl-Before analysis, stock solutions of apo-A-I were diluted with the appropriate molarity of GdmCl in 20 mM NHdHC03, 100 mM NaCI, 0.10 m~ NaN:I, pH 8.0. The solutions were incubated at 23°C for a minimum of 2 h. The results did not change when the incubation was prolonged to 72 h. Reversibility studies were conducted by dialyzing apo-A-I against GdmCl solution in a step-wise manner until the appropriate molarity of GdmCl was reached.

Spectroscopic Studies Circular Dichroism-A Cary model 60 spectropolarimeter with a

6001 circular dichroism attachment (Varian Instrument Division, Palo Alto, Calif.) was used and calibrated with a 0.1% aqueous solution of (d)-10-camphorsulfonic acid. The mean residue ellipticity was calculated as described (12).

Ultraviolet Spectroscopy-A Cary model 219 (Varian) double beam recording spectrophotometer was used. For difference spectroscopy in the presence of GdmCI, the tandem cell arrangement of solvent matching was similar to that described by Herskovits and Laskowski (13). The results were expressed as changes in molar extinction coefficient (Ac,,J.

All spectroscopic measurements were made in thermostatted cell compartments and the temperature was controlled by a Haake water circulating bath (Haake, Inc., Saddle Brooks, N. J.).

~ _ ~ . _ _ _ _ -

Density 1.063 to 1.21 g/ml.

5747

5748 Denaturation Studies of Apo-A-I

Analytical Ultracentrifugation Sedimentation velocity analyses were conducted in a Beckman-

Spinco model E analytical ultracentrifuge equipped with an RTIC temperature control unit and an electronic speed control system. Sedimentation velocity runs were conducted at 52,000 rpm in alumi- num-filled Epon double-sector cells. For dilute protein samples (less than I g/liter), the sedimentation profiles were monitored with the Beckman split-beam photoelectric scanner. The sedimentation rate was calculated from the midpoint of the protein boundary. The schlieren optical system was used for samples with protein concentrations higher than 1 g/liter. Sedimentation profiles were recorded on Kodak metallographic plates and analyzed on a Nikon 6 C microcom- parator (Nikon, Inc., New York). For the analyses of apo-A-I in 2 M and 6 M solutions of GdmCI, a synthetic boundary double sector cell was used at an initial rotor speed of 8,000 rpm until formation of a boundary a t which time the speed was increased to 52,000 rpm to ensure sedimentation of the protein with minimum diffusion. The observed frictional ratio was calculated from

f M(1 - 620)

where so is the sedimentation velocity coefficient at infinite dilution, M is the molecular weight, 6 2 is the partial specific volume of the protein (this parameter was measured in each of the GdmCl solutions studied; see below), p is the density of the solution, N is Avogadro's number, and q is the viscosity of the medium.

Sedimentation equilibrium experiments were performed in a 12- mm Epon double sector cell or a charcoal-filled Epon 6-channel Yphantis cell with sapphire windows. The molecular weight determinations were carried out by both the meniscus-depletion sedimentation equilibrium method of Yphantis (14) and the conventional sedimentation equilibrium techniques as modified by Richards et al. (15) using the initial overspeed period described by Chervenka (16). The studies were carried out in the range between 10,000 and 20,000 rpm and the profiles were monitored with the Beckman split-beam photoelectric scanner.

Viscometry Viscosity measurements were carried out in a Cannon-Manning

semi-micro viscometer (Cannon Instrument Co., State College, Pa.) with a flow time for water of 264 s. In some experiments, a Cannon- Ubbelohde semi-micro dilution viscometer with a flow time of 262 s was utilized. AU measurements were conducted under carefully controlled temperatures in a Cannon model M 1 temperature bath with an external Haake circulator unit. The temperature varied in the range of f 0.005"C. The intrinsic viscosity [q] in milliliters/g was evaluated by solving the following

= [q] + k' [qyc

where c is the protein concentration in grams/ml and specific viscosity v,~, = (q - ~ " ) / q , , , where 7 and q,> are the viscosity of the solution and that of the solvent, respectively. The dimensionless Huggins constant, k' , is a measure of the concentration dependence and considered to be related to the interaction between solute molecules. In general, k' is usually on the order of 10 or higher for associated rigid rods and 0.35 to 2 for linear random coils (17, 18). The axial ratio was estimated from the Simha shape factor (18) as follows

[q] = Y ( 6 > + SI61 + S.,C,,) ( 3 )

where v is the Simha viscosity increment and Ls, is the partial specific volume of apo-A-I in the specified solvent, is the partial specific volume of HsO, 6:1 is grams of GdmCl bound/g of protein, Gr is the partial specific volume of GdmCl at its specified concentration and was obtained from published data (19, 20). The water of hydration S , , in grams/g of protein, was calculated according to the method of Kuntz as described by Lee et al. (21) and was 0.45 g/g of apo-A-I. The Simha shape factor, v, is a function of the axial ratio (a/b) interpreted in terms of a prolate ellipsoidal model (17).

The average dimensions for apo-A-I were calculated from the axial ratio and the following relationship for the volume of an ellipsoid

where a and b are the major and minor semi-axes of a prolate ellipsoid, respectively.

Measurement of Partial Specific Volume

The partial specific volume was calculated from density measurements conducted in a Mettler-Paar (Hightstown, N. J.) magnetic oscillator. The density was measured at 23.0 2 0.005'C. Concentra- tions of apo-A-I were determined from the extinction coefficient at 280 nm which was based on dry weight measurements as described by Kupke and Dorrier (22). From the dry weight measurements and absorption of the diluted stock solution, the extinction coefficient calculated was 1.188 liter/(g cm) a t 280 nm in the buffer and 1.178 liter/(g cm) in 6 M GdmCI.

Preferential interaction and binding parameters in GdmCl were measured and calculated according to Lee and Timasheff (23) as described below. For density determinations a t constant molality of GdmC1, aliquots of apo-A-I (2 to 20 g/liter) were dialyzed against HzO/NH:i, pH 8.5, lyophilized, and then heated in uacuo at 40°C for 48 h. To each dried sample, 1.0 ml of solvent was added and each tube was capped tightly. After incubation for 2 h a t 23°C. the densities were measured. For constant chemical potential conditions, the protein solutions were prepared by dialysis against GdmCl for 3 days at room temperature; equilibrium was reached when the density of dialyzed buffer was within 5 X g/ml of the bulk dialysis solvent. Concentrations for the protein solutions were determined gravimet- rically by diluting the samples immediately after measurement of their densities and reading the absorptions at 280 nm. The apparent partial specific volume c#,.~~ was calculated using the equation (24)

where c is the concentration of protein sample in grams/ml, p is the density of solution in grams/ml, p,, is the density of solvent in grams/ ml. The calculated values of &,,,,, were then plotted as a function of protein concentration and the extrapolated value to infinite dilution was taken as the true partial specific volume (6") .

Column Chromatography

Gel filtration was conducted in glass columns (1.5 X 100 cm) packed with Sephadex G-200 (Pharmacia). The columns were equilibrated with the appropriate buffer with or without specified concentrations of GdmCl and calibrated with known standards: bovine serum albu- min, a-chymotrypsinogen from beef pancreas, whale skeletal muscle myoglobin, cytochrome c from horse heart, and ovalbumin, all pur- chased from Sigma. Blue dextran 2000 (Pharmacia) was used to determine the void volume (VJ and dinitrophenyl-serine for the column volume (VJ. In general, protein solutions were dialyzed for 24 h in the appropriate elution buffer and then applied onto the column. A pump flow rate of 10 ml/h was used and fractions of 1.5 ml were collected into tared test tubes and weighed. The eluate was continuously monitored in an ISCO UA-5 absorbance monitor (ISCO Co., Lincoln, Neb.). The partition coefficient was obtained from (25)

where V,, V,,, and V, are the elution weights of the sample, blue dextran, and dinitrophenyl-serine, respectively. Stokes' radii were calculated by linearization of chromatographic data according to the empirical equation of Ackers (25)

r = a, + b,, erf"(u) (7)

where a,, and b, are column constants and u is defined above. Polyacrylamde gel electrophoresis was carried out in the presence

of either 0.1% sodium dodecyl sulfate or 8 M urea (26). In some cases, protein concentrations were determined according to the Lowry pro- cedure (27), and by amino acid analysis in a Beckman model 121C analyzer.

Chemicals

All chemicals were of reagent grade purity. GdmCl (ultrapure grade) was obtained from Heico (Heico, Inc., Delaware Water Gap, Pa.) and pre-dried in a vacuum desiccator over phosphorous pent- oxide. The presence of absorbing impurities in the GdmCl was

400 nm; only those solutions with less than 0.05 optical density unit checked by ultraviolet absorption spectroscopy in the range of 260 to

Denaturation Studies of Apo-A-I 5749

were used. The concentrations of GdmCl solutions were checked by refractive index measurements using the data of Kielley and Harring- ton (19). All buffers were filtered through sintered glass, and protein solutions were millipored whenever necessary using Millipore fdters which had been pretreated to remove detergents.

RESULTS

Studies on Natiue Apo-A-Z

To assess the behavior of apo-A-I as a function of its concentration in solution and upon perturbation by GdmCI, it was first necessary to make measurements on the protein in its native state under our temperature and buffer conditions. For this purpose, the mean residue ellipticity of native apo-A- I in 20 mM NH4HCO:,, 100 mM NaCI, 0.10 m~ NaNs, pH 8.0, was measured over a more than 500-fold range of protein concentration and the results agreed with those of Osborne and Brewer (1) and Stone and Reynolds (4). Thus, circular dichroic measurements provided a useful diagnostic marker for the dissociation of apo-A-I. On the other hand, ultraviolet spectroscopy in the region between 300 and 250 nm detected no changes down to 3 X IO-'' g/liter of apo-A-I. Only a t this point a small decrease in absorption (-5%) at 280 nm occurred.

Studies on Apo-A-Z in the Presence of GdmCl Spectroscopic Studies-The mean residue ellipticity of apo-

A-I was measured as a function of its concentration in GdmCl solutions. At a protein concentration of 3 g/liter where apo-A- I self-association occurs a biphasic curve was observed (Fig. 1). In contrast, at 0.05 g/liter, where apo-A-I is mostly monomeric, there was only one major sigmoid-shaped curve indicative of a cooperative process. From 0.4 M GdmCl until the end point was reached (see legend to Fig. I), the two curves at 0.05 and 3 g of apo-A-I/liter overlapped with a transition midpoint at 1.08 M GdmC1. This phenomenon was found to be reversible over all concentrations of protein and GdmCl studied (9).

05

(Y

0

0

- E N 10

m al 0 . 5

Q x :: 15 N

T Y

I

20

2 5 0 02 0 4 0 6 O B 1 0 1 2 14 16 18 2(

MOLARITY (GdrnCIl

FIG. 1. Mean residue ellipticity at 222 nm of apo-A-I as a function of the molarity of GdmC1.0, apo-A-I a t a concentration of 0.05 g/liter; 0, at 3 g/liters. The buffer used was as described in text. The measured end point at which apo-A-I is randomly coiled was taken as occurring a t 6 M GdmCl (not shown) where the mean residue ellipticity was -0.016 X lo4 deg cm'/dmol.

Parallel studies monitored by ultraviolet difference spectroscopy gave a pattern representative of the one depicted in Fig. 2. The molar absorptivity (A€,,,) at 285 nm overlaps with that at 292 nm indicative of a denaturation-dependent blue shift in the area of the spectrum corresponding to the tyrosine and tryptophan residues, respectively. A single smooth sig- moidal curve was observed with a transition midpoint at 1.21 M GdmCl even a t a protein concentration of 0.80 g/liter where apo A-I is highly associated. Extrapolation of the straight line portion of the curve to zero concentration of GdmCl gives the A€,,, for the transfer of the buried tyrosine (285 nm) and tryptophan (292 nm) from the interior of apo A-I into the aqueous environment. The extrapolated values obtained cor- respond to five buried tyrosines and two buried tryptophans assuming molar extinction coefficients of 700 and 1600 for tyrosine and tryptophan, respectively (28).

If the system were a two-state transition, i.e. with well defined initial and final states, one would have expected the two curves in Figs. 1 and 2 to have the same transition midpoint. The fact that this was not the case indicates the occurrence of a more complex process.

In order to further define the spectral behavior of apo-A-I, we investigated its hydrodynamic properties.

Studies of Apo A-I in Low GdmCl Molarities (0 to 0.4 M )

The same ultracentrifugal (3) and viscometric procedures (29) that were used in the study of native apo-A-I were applied to the analysis of this apoprotein in GdmC1. First, apo-A-I, 3 g/liter, was studied by sedimentation velocity in various concentrations of GdmC1. As shown in Fig. 3, from 0 to 0.30 M GdmCl apo-A-I was paucidisperse exhibiting both a 2 S and 4 S component. In contrast, at 0.4 M , only a 2 S component. was present indicative of a dissociated state. This difference in association was also observed by molecular sieve chromatography (Fig. 4).

In the presence of buffer without GdmC1, apo-A-I at 3 g/ liter eluted asymmetrically exhibiting at least three maxima, as would be expected for a self-associating scheme, such as monomer-dimer-tetramer-octamer (3, 5). At 0.2 M GdmC1, some dissociation of the apoprotein occurred and at 0.4 M GdmC1, apo-A-I eluted as a symmetrical peak corresponding to a molecular weight of -50,000. For comparison, apo-A-I when chromatographed at 2 and 6 M GdmCl eluted as a single peak with a molecular weight corresponding to that of the

- 4 0

-20-

MOLARITY IGdmCIl

FIG. 2. Representative data of the change in molar absorptivity of apo-A-I as a function of the molarity of GdmC1. The molar absorptivity was measured a t 285 (0) and 292 nm (A) in the concentration range of 0.05 to 3 g/liter. In this instance, difference spectra were obtained against a reference solution without GdmCl containing an identical concentration of apo-A-I (0.799 g/liter) as in the sample measured with GdmCI.

5750

2s 4s

Denaturation Studies of Apo-A-I

OM

0.2M

0.3M

0.4M

TOP FIG. 3. Schlieren patterns of apo-A-I (3 g/liter) in various

molarities of GdmC1. The direction of sedimentation is from left to right as indicated by the arrow. Double sector cells were used with the appropriate buffer as reference. The patterns represent pictures taken 117 min after 2/3 speed was reached.

apo-A-I monomer (28,000 daltons). From the partition and error functions, the calculated Stokes’ radius for the randomly coiled monomer was 48 A. However, although an apparent Stokes’ radius of 31 A could be calculated from the elution profile of apo-A-I in 0.4 M GdmCI, we had to assume that in this case, both apo-A-I and the standard proteins were glob- ular. Such an assumption may not be valid in view of the reported as-ymmetry of apo-A-I (29) as well as the results of this work. For an accurate assessment of the molecular weight of apo-A-I in 0.4 M GdmCl we carried out sedimentation equilibrium analyses. Fig. 5 shows a plot of the apparent weight average molecular weight (Mw. .,,,,) uersus apo-A-I concentration. The Mw,a,,,, (solid line) was calculated from the slope (dlnc/dr’) of the curve at each radial distance according to the equation

2RT dlnc Mw. n,lll =

(1 - C 2 p ) J - Z (8)

where R is the gas constant, r is the radial distance from the center of rotation, T is the absolute temperature, 62 is the partial specific volume of the protein (0.716 ml/g, see below), p is the density of the solution, w is the angular velocity, and c is the protein concentration. Thus, in the presence of 0.4 M GdmCI, apo-A-I was highly but not completely dissociated. The calculated molecular weight of apo-A-I at 3 g/liter in 0.4 M GdmCl was 45,000, a value which is in reasonable agreement with that of 50,000 obtained from the chromatographic studies (Fig. 4). Having established that apo-A-I in the presence of 0.4 M GdmCl dissociates into a new equilibrium state, it was of interest to determine whether changes in shape and/or swelling of the protein had accompanied its interaction with GdmC1. For this purpose, we conducted viscometric measurements on apo-A-I in both its native state and in GdmCl (Fig. 6). In the presence of 0.4 M GdmC1, apo-A-I had an intrinsic viscosity of 8.2 ml/g (Table I) and from sedimentation velocity a frictional ratio of 1.68. These values suggest that in 0.4 M GdmC1, apo-A-I has the same extended structure and asym- metry as the native protein, i.e. the shape of apo-A-I was not significantly affected by the low GdmCl concentration.

+ Vo 45k 25k 178k 124k VI ).

020 0 15 6 M

0 25 0 20 0 15 2 M

VO 45k 12 4k VI + t

E 0 0 5 0

OD N 0.25- O lo:: vo 45k t 2 1 t t t 17& vi

J 020-

5 010-

12 4k

V 0 1 5 - 0.4 M

0 0 5 -

0 2 0 2 5 -

0 15- 0.10- 0 0 5 -

, I

VO 45k 25k 178k VI t

a 020- .( + + +

12 4 k

0.2 M

I

0 2 5 - 0 20-

45k 25k 17& 124k + b t t V, t

0 151 O M

I ’ 50 60 X ) 80 90 1 0 0 110 120 1 3 0 140 150 160

ELUTION WEIGHT (gms) FIG. 4. Gel permeation chromatography of apo-A-I in var-

ious concentrations of GdmC1. The optical density at 280 nm was continuously recorded. V,, refers to the weight of the void volume as determined from the elution position of blue dextran and V, refers to the weight of the column volume according to the elution position of dinitrophenyl-serine. The molecular weight markers and column conditions are as described under “Materials and Methods” and k = 10o0. The conformation of the standards used was either folded (0 to 0.4 M CdmCI) or unfolded (2 and 6 M GdmCI) and reduced and alkylated when appropriate. This was necessary in order to obtain linearity of elution positions for each concentration of GdmCI.

I I I 0.5 1.0 I .s 2 .o

APO A4 CONCENTRATION (011)

FIG. 5. Apparent weight average molecular weight of apo- A-I in 0.4 M GdmC1, pH 8.0. The smooth curve represents a least squares polynomial fit of the average values of Ms. obtained from four sedimentation equilibrium experiments conducted at 23°C.

Studies of Apo-A-Z in High GdmCl Molarities (2 to 6 M) Lee and Timasheff (23) have provided evidence that for

many proteins interacting with GdmCl the number of binding sites can be correlated with the aromatic side chains and peptide bonds. These authors have also shown that the partial specific volume of a protein in denaturing media is a good reflection of a change of its volume and of its interaction with


the solvent components. In order to compare the binding parameters a t low (0.4 M) and high concentrations of GdmCl (2 and 6 M), partial specific volume (U) determinations were carried out at various concentrations of protein and the data extrapolated to infinite dilution. Fig. 7 shows a typical plot of Vapp uersus apo-A-I concentration. In buffer without denaturant, the U values were found to be concentration-dependent. The extrapolated value of 0.729 ml/g is significantly different from that estimated from amino acid analysis (0.734 ml/g). The preferential interaction parameter was calculated according to Casassa and Eisenberg (24) as follows:

(1 - +'2 p0)O = (1 - 4%* POT + (3 (1 - 6 3 p,) (9)

where po is the density of solvent, @'z and +z* are the partial specific volumes at constant chemical potential and constant molality of solvent components, respectively, extrapolated to infinite dilution, & is the partial specific volume of GdmC1, and 53 is the interaction parameter in grams of GdmCl/g of

40 t d

I I L 0 2 4 6 8 IO 12

CONCENTRATION (g/I)

FIG. 6. The specific viscosity as a function of apo-A-I concentration in various molarities of GdmCl as shown. The solid lines were fit to the data obtained by the method of least squares. The viscosity extrapolated to infinite dilution was taken as the intrinsic viscosity.

protein. The change in volume, AV, was calculated from the measured partial specific volume according to the relation

AV = M2(&' - 62) (10)

where M2 is the molecular weight of apo-A-I (28, 171). The amount of apparently bound GdmCl to apo-A-I was calculated from

5 = A.1 - g:jAl (11)

where 5 is the preferential interaction parameter in grams/g of protein, A3 is the amount of GdmCl bound to A-I, g:) is the grams of GdmCl/g of H20, and AI is the absolute hydration calculated to be 0.45 g of H20/g of apo-A-I (21). Table I1 summarizes the data on the binding and volume parameters.

Within the GdmC1 molarities studied, the interactions were preferential with GdmC1. At 6 M GdmC1, the expected and experimental total number of moles bound agreed fairly well, 143 and 181, respectively (23). The observed increase in volume of apo-A-I when transferred from 2 M to 6 M GdmCl is probably a reflection of the increased binding of GdmCl to the protein. This volume change was also indicated, although not as clearly, by the intrinsic viscosity measurements (Fig. 6). By using the observed values of intrinsic viscosity the average end-to-end distance, a>, of apo-A-I was calculated from the relation applicable to a linear random coil (30)

[VIM = @<LZ>"' (12)

0360 1 0.740 - e 0.720

1s 0300 6M I 0 2 4 6 8 IO 12 14 16 18 '

CONCENTRATION (g/ l )

FIG. 7. Apparent partial specific volume. Apparent partial specific volume Cap,, as a function of apo-A-I concentration in buffer without GdmCl (0) and in 6 M GdmCl (A) at constant chemical potential.

TABLE I Apo A-I: hydrodynamic parameters

GdmCl Intrinsic Hugdm Frictional ratio,' Axial ratio,' Stokes' ra- s" X 10'"' viscosity constant, In1 k' f / f n a / b dius," r RG<' Dimension'

ml/g .

A A Low GdmCl molarities

0 2.Oh 9.21 29.76 1.74 (1.48) 6.4 (8) 28 (34) 39 (47) 25 X 164 0.4 2.12 8.20 14.70 1.68 (1.41) 5.7 (7) 31 (33) 38 (44) 27 x 153

High GdmCl molarities 2.0 1.16 23.50 0.63 2.61 (2.12) 12.5 (22) 46 (47) 54 (76) 19 X 265 6.0 0.57 27.64 1.08 2.53 (1.95) 12.0 (20) 48 (49) 57 (82) 18 X 284

end dis- End-to-

tance' A

107 103

147 155

Sedimentation coefficient extrapolated to zero protein concentra- profile (Fig, 4). The radii in parentheses were obtained according to tion.

The frictional ratio was calculated from Equation 1. The values in parentheses represent the effect of shape alone on the frictional coefficient expressed as

an equivalent sphere model as (IS) 10rN 3M

[VI = - t3R03 and r = ERG

where Rc is the radius of gyration and 5 is a constant equal to 0.875. The values in parentheses refer to a rod-shaped model where Rc;

is given as where 6, = 0.45 g of HzO/g of protein.

The axial ratio, a/b. was calculated from the intrinsic viscosity, each corresponding value in parentheses refers to that calculated from the frictional ratio, f / f shape. A prolate ellipsoid was assumed as where = 2a.

where apo-A-I is a random coil. the equivalent shape, which would not be valid a t 2 and 6 M GdmCl /The dimensions were calculated from the axial ratio and ti^^

coefficients according to Equation 7. The value of native apo-A-I (0 ing a linear random coil. M GdmC1) was obtained from the partition coefficient corresponding k Ref. 29. to the Lowest molecular weight component (-35,000) in the elution

L Z RC2 = -

12

The Stokes' radius was calculated from chromatographic partition The end-to-end distance was calculated from Equation 12, assum- 4 assuming a prolate ellipsoid.


TABLE I1 Apo-A-I: partial specific volumes and preferential interaction parameters

GdrnCl mo- "';" larity +:: 4::: I" GdmC1'

Bound A V'

mI/g ml/g m I/g R/R mol/mol mi/mol -

0.0 0.729 f 0.002 0.4 0.736 T 0.002 0.716 k 0.002 0.07 f 0.02 27 2.0

197 T 56 0.735 f 0.002 0.711 f. 0.002 0.11 r 0.02 63

6.0 169 2 56

0.736 f 0.001 0.718 f 0.002 0.16 f 0.02 181 197 k 48 n - 0

0 0 vq , partial specific volume at infinite dilution. &,, partial specific volume at infinite dilution under conditions of constant molality of solvent components.

.$, preferential interaction parameter. Assuming 0.45 g of H,O/g of protein.

' R , , partial specific volume at infinite dilution under conditions of constant chemical potential.

'AV, change in volume due to transfer of native protein to denaturing medium.

; zoo 400 600 800

RT Inbkaf

FIG. 8. AGn as a function of RTln(1 + ka) where k = 0.8 and a is the molarity of GdmCI. 0, data obtained from circular dichroic measurements (An = 9.8, AG/,H?" = 3.70 kcal/mol); 0, from ultraviolet difference spectra (An = 10.33, AG/I"~(' = 4.08 kcal/mol).

where [ q ] is in milliters/g, M is the molecular weight of apo- A-I, and Q, is an approximate universal constant equal to 2.1 X lozf3. The hydrodynamic data obtained on apo-A-I are summarized in Table I.

Estimation of Free Energy We next attempted to estimate the free energy of stabili-

zation of apo-A-I in aqueous media by taking into account the notion derived from the studies outlined above that the denaturation of monomeric apo-A-I does not obey a two-state transition and thus is not easily amenable to a precise math- ematical description. The standard free energy of unfolding, AGu, was calculated in terms of a simple equilibrium N F? D where N is the native and D the unfolded form of the protein, assuming that intermediate species were not present in appre- ciable concentration a t any stage of the transition. On this assumption values of equilibrium constants, KI,, between 0.1 and 10 were obtained from the data in Figs. 1 and 2, and from the relation

KI, = - Y - Y,, Yl, - Y

where Y is the observed parameter at any concentration of GdmCl and Y,v and YI, are the values for the native monomer and for the unfolded product. Since, as shown above, apo-A- I dissociates at 0.4 M GdmC1, the mean residue ellipticity was extrapolated from 0.4 M to 0 M GdmCl to obtain YN. Moreover, since the change in molar extinction was independent of protein concentration 'we obtained the values of Y N by direct extrapolation as shown in Fig, 2. The free energy of denatur-

ation was then determined from the experimental data using the relation: PGI, = -RTln K , , where R is the gas constant and T the absolute temperature. The smooth curves in Fig. 8 were fit by the method of least squares to the equation based on denaturation binding (31)

AGI, = AGr,H20 - AnRTln(1 + ka) (14)

where A G I I ~ ~ " is the free energy in the absence of denaturant when a is in terms of molarity of GdmCl and An is the difference in the number of GdmCl bound by the protein in the denatured and native states.

DISCUSSION

Our results have shown that the process of denaturation of human apo-A-I by GdmCl is strongly dependent upon the concentration in solution of both apoprotein and denaturant. Based upon the results of the several physical measurements carried out in this work, this process of denaturation can be divided into those events occurring a t low molarities of GdmC1, mainly a reflection of apo-A-I oligomer dissociation, and those occurring at moderate to high concentrations of GdmC1, mainly a reflection of the unfolding of the dissociated apo-A-I monomers.

Denaturation ofApo-A-I in Low Molarities of GdmCl(0 to 0.4 M)-As assessed by circular dichroic measurements (Fig. l), sedimentation velocity (Fig. 3), and molecular sieve chromatography (Fig. 4), apo-A-I, in the presence of GdmCl (0.4 M) undergoes important structural changes. In addition, the sedimentation equilibrium results (Fig. 5) reveal that apo-A- I acquires a new equilibrium state, which in denaturant-free aqueous buffers was previously shown to conform with a monomer-dimer-tetramer-octamer model ( 3 ) . This process of dissociation appears to be related to the preferential interaction of the apoprotein with GdmCl as indicated by the changes in partial specific volume of the apoprotein when measured a t constant chemical potential and constant molality of GdmC1. That the changes attending the transfer of apo-A-I from 0 to 0.4 M GdmCl were due to a dissociative process is also indicated by the decrease in the value of the Huggins constant which was calculated from the viscometric data (see Table I): at 0 and 0.4 M GdmCl the intrinsic viscosity and thus the shape of apo-A-I, exhibited no significant changes whereas specific viscosity uersus apo-A-I concentration plots showed modifications in slope (Fig. 6), which are compatible with a dissociative process. This lack of shape change of apo-A-I a t low molarities of GdmCl is also supported by the ultracen$ri- fuga1 measurements and particularly by the value of the frictional ratio (Table I ) which remained essentially un- changed with respect to the value of the native state. Thus, low molarities of GdmCl appear to influence the state of oligomerization of apo-A-I but induce no significant changes in the conformation of this apoprotein.


Denaturation of Apo-A-I in High Molarities of GdmCl (2 to 6 M)--In 2 M GdmC1, the circular dichroic measurements together with the ultracentrifugal and viscometric studies could be interpreted to indicate that apo-A-I had acquired a random coil structure. However, upon increasing the GdmCl concentration to 6 M , further changes in the structure of apo- A-I took place as indicated by the following: (1) a decrease in mean residue ellipticity, (2) a decrease in the sedimentation coefficient, (3) an increase in the intrinsic viscosity, and (4) a change in the partial specific volume. According to Tanford et al. (32) the relationship between n, the number of residues in a randomly coiled polypeptide chain and the intrinsic viscosity of the latter, can be expressed as [q] = 0.684n"". When this relation is applied to apo-A-I, the calculated value of the intrinsic viscosity, 27.1 ml/g, compares satisfactorily to the experimental one in 6 M GdmC1, 27.64 ml/g. Thus, it appears that apo-A-I in 6 M GdmCl is fully denatured. The structural differences of apo-A-I between 2 M and 6 M GdmCl may be an expression of the marked difference in the number of GdmCl molecules bound to this apoprotein (see Table 11). Interest- ingly, the transfer of apo-A-I from 2 to 6 M GdmCl was attended by an increase, although small, 8 A, of the end-to- end distance of the polypeptide chain (see Table I), probably the result of restrictions in rotational motion imposed by the GdmCl molecules on their interactions with the random coil.

Zntegrated Information from the Studies at Low and High GdmCl Concentrations-An interesting observation resulting from our studies is that the length of apo-A-I as a rod-shaped molecule in low GdmCl molarity was comparable to that of a random coil in a highly denatured state. In either its native state or in low molarities of GdmC1, apo-A-I exhibited sedi- mentaiton velocity and viscosity data which are compatible with an asymmetric rod, having an average axial ratio of 6.0 (Table I). Based on the fact that apo-A-I is highly helical, one could envisage this molecule as folded upon itself (29) (diam- eter of 26 A, twice that of an a helix) with a long axis of 150 to 160 A. On the other hand, in 6 M GdmCl where this apoprotein is a random coil, the end-to-end dimensions, 155 A, may be accounted for by a coil having rotational restrictions as a consequence of its interaction with GdmC1. These restrictions would expand the length of this coil, which in an unre- stricted state would be only 82.5 A long (30). Thus, we can envisage apo-A-I as a helical rod when in its native or slightly denatured state, become a coil of a comparable length but of increased volume as a consequence of its unwinding and expansion in high concentrations of GdmC1.

In regard to the energetics of the helix-random coil transition attending the action of GdmC1, a few comments are in order. The lack of overlap between circular dichroism and ultraviolet spectroscopic data in GdmCl suggests that the exposure of the aromatic residues, tyrosine and tryptophan, occurred independently of the unfolding of all or portions of the helices in apo-A-I, and that the denaturation process of monomeric apo-A-I did not obey a two-state N s D transition (30) in agreement with the results of Gwynne et al. (7) and Reynolds (9). In addition, according to our data, the exposure of the aromatic amino acids was only dependent on the molarity of GdmCl and not on apo-A-I concentration. It must be noted that the values of the free energy changes were 3.70 and 4.08 kcal/mol, respectively, values which are considerably lower than those reported for most proteins: myoglobin and lysozyme, 9 kcal/mol; ribonuclease, 16.1 kcal/mol; and a-chy- motrypsin, 11.9 kcal/mol (33). This low free energy change suggests that apo-A-I in its monomeric form has an unstable folded structure in solution which is in agreement with the results of Reynolds (9). Since the energy which is required for both dissociation and unfolding, should a priori be much

greater than that relating to the unfolding process alone, the associated form of apo-A-I is expected to give structural stability to the protein in solution. It should be noted that the value of 2.7 kcal/mol which was repoljed by Tall et ~ l . (10) for the stabilization of the native conformation of apo-A-I is lower than that found in our present work. However, those authors monitored the conformational changes by calorime- try, a technique which is not as sensitive as circular dichroism in detecting secondary structural changes in a protein. More- over, some studies were carried out in urea solutions and monitored by ultraviolet difference spectroscopy, conditions which both have limitations; in one case the urea was not shown to induce complete denaturation of apo-A-I; in the other case, we found that ultraviolet spectroscopy is unable to detect concentration-dependent changes.

With regard to binding sites for GdmC1, the number which we calculated from the measurements of partial specific volumes for the transfer of apo-A-I from 0.4 M to 6 M GdmCl is significantly different from the value of 9.8 obtained from the circular dichroic data (see Fig. 8 and Tables I and 11). This discrepancy can perhaps be reconciled if one considers that binding sites as defined in Equation 14 may also refer to binding domains (31), i e . regions where more concentrated GdmCl is nearer to the protein than to the bulk solution. It is possible that these postulated binding domains are identifiable with the actual number of helices, 8 to 10, predicted for apo- A-I (2, 34-37). Since binding sites for guanidinium ions are much more likely to be available in the unfolded portions of a polypeptide chain than in a folded region of a molecule (38), the increase in bound GdmCl that we observed when apo-A- I was transferred from 0.4 M GdmCl (folded apo-A-I monomer) to 2 M GdmCl (unfolded monomer) and to 6 M GdmCl (random coil) is not surprising. This interpretation is compatible with the notion that GdmC1, once introduced into the nonpolar and polar domains of a protein stabilizes those areas which are exposed to the solvent. In this solubilization process, the absolute numbers of guanidinium ions which are actually bound to the protein may not be as important as the relative changes in the GdmCl binding attending protein unfolding. The latter information, which is expected to give insight into the actual mechanism of denaturation may be obtained from kinetic studies.

When taken together, all of our results provide interesting information on the structure of apo-A-I in solution. First, apo- A-I is asymmetric in both its associated and dissociated form. Second, the monomer, although having highly helical rigid domains, may be more appropriately considered as a helical coil (39) with flexible interconnecting P-bends. Third, apo-A- I must have little or no stable tertiary structure as reflected by its very low AG,jHJ". Based on these notions, studies on the interaction of apo-A-I with lipids or with a lipoprotein such as high density lipoprotein must be considered in the context of competitive reactions among intra- and interchain helices and among helices and lipids. For instance, an energy barrier preventing the interaction of apo-A-I with lipids can be as- cribed to the preference of apo-A-I to associate with itself. In past studies, we have stressed the need for defining the state of the association of apo-A-I in solution before ligand binding studies are undertaken. It is now apparent that greater restrictions apply in regard to the conformational state of the apo- A-I monomer. These considerations appear even more com- pelling when protein-ligand binding studies are to be carried out in the presence of a denaturant such as GdmCl because of the difficulties of dissecting its effects on the protein, on the ligand, and on the interactions between them.

Acknowledgments-We wish to thank Miss Naomi Sakoda and


Miss Maria Garcia for valuable assistance; Mr. Don Barbeau and Dr. Gunther Fless for providing helpful discussions during the ultracentrifugal studies.

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effect of guanidine hydrochloride on the hydrodynamic and ... · that of the solvent, respectively....

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