Concanavalin. A: A Stopped Flow Nuclear Magnetic Resonance .... in the magnet gap. A 36.inch ... water resonance where Z( m) = intensity at time infinity, 6 =.
Vol.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 250, No. 5, Issue of March 10, PP. 1618-1624,
Printed
1975
in U.S.A.
Concanavalin A: A Stopped Flow Study of Conformational Changes and a-Methyl-D -Mannoside*
Nuclear Induced
Magnetic Resonance by Mn++, Ca++,
(Received for publication, JOHN J. GRIMALDI From
AND BRIAN
the Department
of Chemistry,
D.
July 26, 1974)
SYKES
Harvard Universit!y,
SUMMARY
Cambridge,
Massachusetts
02138
EXPERIMENTAL
Concanavalin h is a phytohemagglutinin isolated from the jack bean which displays a range of properties associated with its ability to bind to cell surfaces (1). Physical studies of this protein indicate that in the pH range 4.5 to 5.6 Con A’ exists as a dimer of molecular weight 55,000 composed of 2 identical monomeric units (2, 3). The protein has been reported to bind 2 metal ions per monomer in an ordcrcd fashion (4). First, a transition metal ion is bound in a site designated Sl and then a calcium ion can bind (site SZ). Both metal ions, in turn, influence the
binding of simple sugars such as cY-methyl-u-glucoside (4). Earber and Carver (5) have observed a time-dependent change in the proton relaxation enhancement of water when AIn++ was added to a solution of Con A. Other workers have also observed time-dependent changes in the carbohydrate binding ability of the protein after addition of AIn++ and Ca++ to demctalized Con A (6). The application of ShlR to monitoring rapid biochemical reactions and detecting intermediates has recently been extcndcd, with the development of rapid mixing devices within the NXR probe, into the stopped fiow range (7). In this paper we report the application of stopped llow NAlR to the elucidation of the mechanism of the conformational changes induced in concariavalin h by i\In ++, Ca++, and simple sugars such as cY-methyl-u-mannosidc. The advantage of NLIR as an ob* This work was supported by National. Institutes of Health Grant GM-17190 (B. I). S.) and Training Grant Fellowship (J. J. G.) and an Alfred P. Sloan Foundation Fellowship (B. D. S.) 1 The abbreviations used arc: Con A, concanavalin A; olMM, a-methyl-o-mannoside. 1618
PROCEDURE
Con A was purchased from Calbiochem (Lot No. 210073) in lyophilized form. Demetallized protein was prepared by the method of Kalb and Levitzki (4). Protein (250 mg) was dissolved in 30 ml of 1 M NaCl and t,he pH lowered to 1.2 using 1 M HCl. After 45 min, the protein was transferred to a dialysis bag and dialyzed against three changes of 1 liter each of distilled water over a S-hour period. The protein solution was then checked for Mn and Ca using a Perkirl-Elmer model 303 atomic absorption machine. The protein solution was then lyophilized and dissolved in the appropriate buffer. Protein concentrations were determined spectrophotometrically at 280 nm using Eiz,,, = 11.4 at pH 5.6 (8). Buffer solutions were 0.05 M sodium acetate, 0.2 M NaCl adjusted to pH 5.28. Mn++ and Ca++ solutions were prepared by adding appropriate volumes of stock MnClz and CaClz to aliquots of buffer. cu.Methyl-D-mannoside (Schwarz-Mann) was weighed out and buffer added to obtain the desired concentration. All measurements were made on a 40.5 MHz stopped flow NMR spectrometer. The flow system consists of a rapid mixing cell and driving and stopping syringe blocks. The syringe blocks were made from nylotron with rexolite valve keys. Ijriving syringes are ordinary 5.ml B & D glass luer-tip syringes that were epoxied int,o threaded rexolite sleeves which screwed into the syringe block with O-ring seals. The stopping syringe was identical except a 2.ml size was used. The driving syringes were actuated by a solenoid valve-controlled air piston. The mixing cell consists of three parts (Fig. 1). The top portion contains two premagnetization reservoirs, one for each reactant. E:ach reservoir is const,rictcd and divided into two small diameter jets. These four jets empty into an eight-jet mixer similar in design to that of Gibson (9). From the mixer, the reactants flow into an observation tube (3 mm, inside diameter) which has a receiver coil wrapped around it. The third piece is a continuation of the observation tube with a >&inch rigid plastic tube epoxied t,o the bottom. In addition, a malt BNC connector with the center pin drilled out is epoxied to the bottom with the exit tube running through it,. The three parts were connected via O-ring seals. Connections to the syringe blocks were made with nylon Swagelok connectors via small lengths of rigid plastic tubing and thick walled glass tubing. To accommodate the cell, the V-4331 probe had to be modified. The top plate of the probe was enlarged to t,he diameter of the Faraday shield. In addit,ion, an aluminum block was mounted on the plate to allow the probe to be oriented at 90” to it,s normal orientation in the magnet gap. A 36.inch hole was drilled through the female BNC connector and cover plate at
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servation technique for transient kinetic experiments is that the SilIR parameters determined for the species involved in the reaction can be interpreted in terms of local molecular parameters such as number of ligand sites, ligand exchange rates, and correlation times for the interaction.
The conformational changes induced in concanavalin A by the binding of Mn ++, Ca++, and oc-methyl-D-mannoside have been studied at pH 5.28 by stopped flow nuclear magnetic resonance techniques. Three distinct conformation states of the protein have been kinetically observed and an ordered binding mechanism elucidated from a detailed analysis of the reaction records. In addition, the individual steps of this mechanism are interpreted in terms of molecular parameters characterizing the conformational states involved such as ligand exchange rates to the paramagnetic Mn++.
1619 was varied. The reaction records were analyzed for first order kinetics by using the previously derived expression (7) I(t)
I (-)
=
(1)
(1+6e-kohst)
for
Recewer Coil
time
dependence
of the intensity
of the
absorption
mode
---
Extt
Tube
0
I.
(d)
cu
1. Diagram of the three components of the stopped flow NMR mixing cell (not drawn to scale). The top portior~ shows the reservoirs and delivery jets. The middle portiotl shows the mixerobservation tube with the receiver coil. The bottom portion shows the exit section with incorporation of the BNC connector and rigid plastic tubing (see text). The four circular figures to the right arc cross-sections of the cell at the points indicated by the arrowa and show the relative orientations of the t.hree pieces when assembled. Section a shows the four delivery jets and b shows the four grooves which feed the eight-jet mixer. c and d illustrate the two four-jet tangential mixers. When the cell is assembled, the four delivery jets are oriented 45” to the four grooves of the mixer. This initiates mixing prior to the eight-jet stage. FIG.
+t + Ca
kl
0
II.
1
k-l
kl
i-t a 1/
K2
+ Ca
In the limit that [Ca++] >> [Con
was accomplished
and
a Biomation
GlOB
transient
recorder
inter-
RESULTS
Conjormational Changes induced by Ca++ and JIn++-These experiments consisted of mixing a buffered solution of demetallized Con A with a similarly buffered solution of XlnClp and CaC12. I>uring each series of experiments the concentrations of Con A and Mn++ were fixed whereas the concentration of Ca++
Z/
k
ohs
=
kel
+
kl[ca
++l
n
Ca
II
MECHANISM
The flow transient and permitted monitoring smaller linewidths. spectrometer was interfaced to a Nova 1220 12K computer with a computer operations LINC tape drive and a Megatek DA converter providing scope display and hard copy. AD conversion with
a -A
I
MECHANISM
Since the the bottom of the probe to accommodate the exit tube. center pins of the BNC connectors were removed, the live side of the receiver coil was soldered directly to the tuning capacitors. The ground was made in the usual way. The spectrometer is similar to that previously described (7) with the following modifications. First, a lock channel was incorporated. This prevented drift during the acquisition of a stopped
faced to the computer. During a typical experiment, the spectrometer remained locked on water, the driving syringes were actuated, and the resulting transient was recorded by the Biomation GIOB. Subsequently, the data was transferred to the computer and stored on LINC tape for future analysis.
-k-l
A],
for kechanism I
(2)
where KS is the dissociation constant for the second step which is assumed to be in rapid equilibrium (11). Equation 1 predicts a linearly increasing kOb8 with increasing Ca++ concentration, and Equation 2 a decreasing koas with increasing Ca++ concentration; neither are in agreement with the observed results. The following mechanism is consistent with the data.
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BNC Connector
the
water resonance where Z( m) = intensity at time infinity, 6 = (Z(m) - Z(O))/Z(O) = amplitude change, and kobs = observed first order rate constant. These three parameters were varied to produce the best nonlinear least squares fit to the data. The Ca++ dependence of kobs, 6, and Z(m) are shown graphically in Figs. 2, and 3, A and B, respectively, for [Con A] = 0.14 mM sites and [Xln++] = 0.5 InM at pH 5.28. (Unless specifically stated otherwise all concentrations refer to concentrations after mixing.) Assuming a dissociation constant for Mn++ of 50 pbf (lo), this corresponds to about 90% saturation of the protein Xln++ binding sites. Since the bimolecular binding of Mn++ to Con A is assumed to be rapid on the time scale of these stopped flow measurements, most of the protein will be bound with &In++ at time zero. In the following discussion of possible mechanisms which are consistent with our results, we will represent different conformations of the protein-Plln++ complex, as identified by their different linewidths, by geometrical figures. On the basis of the dependence of kobs on the concentration of Ca++, the following two mechanisms can be eliminated:
1620
CP
II
k-l
kl 41'
III
5
,k-2
Ca
k2
where K1 is the dissociation constant describing the rapid preequilibrium binding (see Ref. 12 for derivation of rate equations). kobs increases up to a plateau value with increasing [Ca++] in agrecmcnt with Fig. 2. In addition, L-2 must be small relative to ke/(l + KJ[Ca++]) since the final intensity readings are constant at Ca++ concentrations low with respect to K1. Extending Mechanism III to include an additional dissociation step (see below and Fig. 4),
+
+
Ca*
Ca++
(8) .
. l
.
.
. .
.
c 5
7 I-
210
i
I
I
5
K3
q Ca
k2 A Ca
MECHANISM
final intensity
enhancement
=75 6
I
7473. 72-
A
0 K1II
relaxation
mM
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i 0
the constant
25
(4)
= k-2 + (I I;&)
2
IV.
20
FIN. 2. A plot of the observed rate constant kohs, as a function of Ca++ concentration. Each value of k,,+ was obtained by mixing demetallized Con A ( [Con A] kohs
I5
[Co"]
?A
III
MECHANISM
IO
Ca
readings
of the A
IV also imply that the proton state is identical
with that
of A Ca state. This conclusion is based on the estimated fraction of Ca++ sites bound at equilibrium. Using a binding constant of 300 pm (4), the fraction of sites occupied by Ca++ are 0.75 and 0.96 for total [Ca++] of 1 and 25 mM, respectively. To understand the [Ca++] dependence of the amplitude (Fig. 3A), it is necessary to know the time dependence of all of the species that contribute to the linewidth. The total linewidth is the fast exchanged average of the contributions of all of the sites
IO
15 [Co++]
20
25
mM
3. Ca++ concentration dependence of the amplitude change 6, (A) and final intensitv I cm) (Bl. 6 and Z (M j were obtained bv n%ing demetallized Co; A’(