[47] Use of hydrogen exchange kinetics in the study of the dynamic properties of biological membranes

630

PROTONSAND MEMBRANEFUNCTIONS

[47]

[47] Use of Hydrogen Exchange Kinetics in the Study of the Dynamic Properties of Biological Membranes By

ANDREAS

ROSENBERG

Introduction to the Principles of Hydrogen Exchange Kinetics The basis for the usefulness of hydrogen exchange kinetics in the study of conformational states of macromolecules lies in the observation that the rate of exchange from peptide groups and similar N - H and O - H bonds is extremely sensitive to the conformational state of the whole molecule. RR'CONH

+ HOEH ~ - R R ' - C O N 2 H + H O H

(1)

The difference in the rate of exchange from a peptide bond in the native, folded state and the corresponding unfolded state can reach l0 orders of magnitude, providing there are very favorable conditions for resolving effects due to different types of conformational motion. The desired information for any single exchanging site comes in the form of an attenuation factor fl included in the apparent rate constant for exchange flkex = kapp. The constant k~x stands for the rate constant for exchange from the same site if all conformational restrictions are removed, a state often referred to as the unfolded, random coil. An ideal random coil state is difficult to achieve in practice and as a rule the rate constant is assumed to be very similar to the rate constants of short peptides of similar amino acid composition. The practice of hydrogen exchange has two sides: (1) gathering of kinetic data that, in principle is not too complicated, but demands considerable laboratory skill and patience; and (2) extraction of reasonable estimates for the factor/3 and interpretation of the results in terms of conformational motion. Without doubt, it is the second aspect that has been the most difficult. It is also in this area where the most prominent advances have taken place during the past few years. The full scope of the method, including experimental methods, data handling, and interpretation, are treated in another chapter of this publication.~ In this chapter, both advantages and disadvantages of the method are reviewed as applied to systems such as membranes and other cellular particles, systems quite different from dilute solutions of a single low-molecularI R. B. G r e g o r y a n d A. R o s e n b e r g , t h i s s e r i e s , Vol. 131 [21], in p r e s s 0 9 8 6 ) .

METHODS IN ENZYMOLOGY, VOL. 127

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

[47]

HYDROGEN EXCHANGE KINETICS

631

weight protein, the study of which has been the goal of most hydrogen exchange practitioners. 2-4 In the case of membrane-bound particles, one has to remember that the attenuation factor/3 has two components: one due to the intrinsic structure of the protein with its hydrogen bonding system and patterns of recurring secondary structural features; the second due to the interactions of the protein either with the lipid bilayer, the proteins of the cytoskeleton, or with the more complicated structures present in the cell. These factors are not necessarily independent and their separation is often difficult. The complexity of the system to be studied makes the use of some of the more powerful techniques of exchange such as nuclear magnetic resonance (NMR) and neutron diffraction not very promising. The presence of a large amount and variety of constituents tends to produce a background of such complexity as to swamp signals from a single protein molecule. This focuses our attention on methods of lesser resolving power, but with less sensitivity to background signals. Methods such as tritium trace label and infrared (IR) spectroscopy as a rule provide information about the exchange taking place simultaneously from many independent sites. One is dealing thus with distributions of rate constants instead of isolated single rate constants. These studies have recently received a boost by development of mathematical methods able to handle the distribution function of rate constants and extract information on a level comparable to that obtained from the study of single sites. 5 Despite the limitations in choice of the method to use for membrane studies, the method most suited, the tracer labeling method, provides inherent advantages for the study of complex nonhomogeneous systems. By selectively labeling one isolated protein and then reinserting it into the complex or membrane, we are able to follow the behavior of just this single protein without any background, although the ratio of labeled protein to unlabeled species of the complex may well be 1 : 100. In that sense, the method is very similar to spin-label carrying reporter groups. However, the observed behavior is dominated by the sum of changes and motions in the whole protein molecule, in contrast to the immediate surroundings of a single site, mirrored in spin-label studies. Another advantage of the radioactive tracer method, utilizing tritium, is our ability to provide a second permanent label in the form of 14C for the protein. This A. 3 S. 4 C. 5 R.

D. W. K. B.

Barksdale and A. Rosenberg0 Methods Biochem. Anal. 28, 1 (1982). Englander and N. R. Kallenbach, Q. Rev. Biophys. 16, 521 (1984). Woodward and B. D. Hilton, Annu. Rev. Biophys. Bioeng. 8, 99 (1979). Gregory, Biopolymers 22, 895 (1983).

632

PROTONS AND MEMBRANE FUNCTIONS

[47]

label acts as a concentration marker which makes the experiment independent of the sample size isolated. The IR methodology is relatively insensitive to the homogeneity of the system and it is little influenced by light scattering, allowing for analysis of particulate matter deposited on surfaces. The tritium method is totally independent of the nature of the sample. Cell constituents and fragments of any type can be studied once a labeled protein has been inserted or a successful labeling in situ has been carried out. A problem associated with the use of hydrogen exchange kinetics is the paramount importance of the definition of hydrogen and hydroxyl ion activities in the sample to be investigated. The exchange mechanism is catalyzed both by hydrogen and hydroxyl ions with direct catalysis of water playing an increasing role at higher temperatures.l Thus, a small shift in hydrogen ion concentration produces considerable shifts in the exchange rates. Effects observed are similar to those produced by changes in the dynamics of the conformation. Changes in the apparent rate constant kapp can be due both to changes in/3 and in kex, the latter being a direct function of hydrogen and hydroxyl ion concentration. It is imperative therefore that the hydrogen ion activity be kept constant and its value determined. Uncertainties about pH of the sample make an interpretation of the results impossible. Preferred Methods for Data Gathering Methods such as NMR, neutron diffraction, and UV are not suitable for the study of large complexes within cellular architecture. These methods are described in detail in another chapter of this publication.~ The methods described below represent, in our opinion, the best approach at this point in time and have, as a rule, been used for the study of membrane constituents. The methods fall into two classes, depending on the nature of the isotope used. Tritium exchange methods include rapid filtration and fast dialysis techniques, whereas deuterium methods rely on infrared spectroscopy. Tritium Methods

First, a general description of the tritium labeling methodology is presented covering common aspects for both filtration and rapid dialysis methods. The kinetics studied in tritium methodology represent, as a rule, out-exchange, i.e., loss of radioactive label from a molecule that has previously been brought to isotope equilibrium with a solution containing tritium in the form of tritiated water molecules. As the first step of the out-

[47]

HYDROGEN EXCHANGE KINETICS

633

exchange, the excess tritium in the solution is removed rapidly, either by filtration or fast dialysis. The loss of label that takes place after that, at controlled conditions, represents the desired out-exchange reaction. Because of the removal of excess tritium, the back-exchange during the experiment can be discounted and the out-exchange from any single site will be of the first order. In-Exchange. In-exchange, the first step of the experiment, has to take place at such a pH and temperature as to assure that the isotope equilibrium will be reached in reasonable time. Because OH catalyzes the exchange more efficiently than the hydrogen ion, 2 it is customary to inexchange at as high a pH and temperature as possible. The limits are set by protein stability and vary from protein to protein. The presence of isotope equilibrium is determined kinetically by measuring the level of incorporation as a function of in-exchange conditions. Time is the most common variable. If no further change is observed after incubation time is doubled, the in-exchange solution is considered to be at equilibrium. Although the conclusion has a high probability of being true, there can be exceptions especially with thermally very stable proteins. 6 If one uses a temperature of 35° for in-exchange of a protein with Tm of 50° at this pH, one will get better in-exchange than in the case for a protein with Tm = 8 0 °.

The degree of tritium labeling achieved is determined by the relationship between np and ns, which represent the total number of sites available for the isotope on protein and solvent molecules. Thus np/n s =

np/ns

(2)

where the asterisk notes the concentrations of the tritium isotope. The usual value for n~ in dilute aqueous solution is 111 mol. In order to compare that with the np for a 5% solution of a 30 kDa protein, one uses an approximate estimate of np 0.7. From these estimates, one sees that if we want to count the label for the last 1% of the exchanging proteins with precision, we have to take account of the dilution factor of 104 in choosing the dose of tritium for in-exchange. Consequently, the level of radioactivity of the in-exchange solution varies usually between 0.5 mCi to 0.5 Ci, depending on the amounts of protein available. At high levels of radioactivity, great care must be taken in handling the in-exchange step. The removal of excess tritium must take place in a well-ventilated hood. The upper limit for the radioactivity used is determined by the necessity of retaining the conditions of trace labeling n~/np <0.01. As an example for in-exchange, these conditions can be used for the study of hemoglo=

6 H. B. Osborne,

FEBS Lett. 67, 23 (1976).

634

PROTONS

AND MEMBRANE

FUNCTIONS

[47]

bin: 50 mg of protein in 1 ml o f p H 9.2 buffer (pH can also be adjusted with 0.1 N NaOH) is mixed with an equal volume of tritiated water to give a final activity of 0.4 mCi. Incubation time is 24 hr at 32 - 0.5 °. Out-Exchange. Once the isotope eqilibrium has been reached, excess label is removed by either filtration or rapid dialysis (see below). The sample is redissolved or buffer changed to provide the desired conditions for out-exchange. The exchange is followed by removing small samples at desired time intervals. The procedure for removal of excess label is repeated for each sample (in rapid dialysis, it is accomplished in a continuous fashion). The residual radioactivity is counted using any commercial scintillation cocktail. We have had good results when including 10% Beckman Biosolve BBS-3 into our toluene-based cocktail. The data will appear as pairs of time and DPM (correction for quenching and counting efficiency to be carried out according to the procedures recommended for the instrument used in counting). Now, let us relate CPM(t) to H(t). For in-exchange one assumes that isotope equilibrium has been reached, thus the label will be distributed according to the number of available sites. Let np be the number of sites on a protein molecule, P be the concentration of protein in in-exchange solution, CPMp represents counts attributable to tritium on the protein, and CPMo represents the counts due to solvent. One can write, assuming a molarity of 111 for water protons,

Pnp

_

CPMp

(3)

CPMo

111

The number of sites on protein is, as a rule, 103-104 times less than the number provided by the solvent, water, so CPMo can be equated with the total count seen in in-exchange solution. We should remember that CPM and CPMo refer to count per volume unit of in-exchange solution, so that the dilution factors due to sampling should appear in calculations. At any time point along the exchange curve the sites remaining labeled H(t) are represented by a count CPM(t). One can write

H(t)

np

CPM(t) -

-

-

CPMp

(4)

Using the previous equation for np, one arrives at the standard equation used in calculations

H(t) -

CPM(t) 111 P CPMo

(5)

P refers now to protein concentration in the solution sampled. The detailed derivation presented is useful to highlight the simplifications used

[47]

HYDROGEN EXCHANGE KINETICS

635

in arriving at Eq. (5). In highly concentrated solutions of high-molecularweight material, the molar concentration of water protons may be less than 111 because of excluded volume effects. Further, the rate observed reflects the loss the tritium and is different from the rate of hydrogen loss due to the kinetic isotope effect. In using Eq. (5) to calculate an absolute number of exchangeable hydrogens, keep in mind that derivation of Eq. (5) was based on the concept of isotope equilibrium [Eq. (2)]. In peptides and randomly coiled polypeptides, tritium has a greater affinity for peptide sites than for water sites (isotopic enrichment). Thus, for peptides or randomly coiled polypeptides, the calculation of H(t) should include an isotopic enrichment factor of about 1.2 if the calculated H(t) is to represent an absolute number of hydrogens. The isotope effect in proteins is more complex. 2 Equation (5) contains the variable P standing for protein concentration in the sample used for counting. It has to be determined with equal precision to counting the radioactive label. For the study of dilute solution of a single protein many methods are available, measurement of UV absorbance being the most common. In more complex situations where the labeled protein represents but a fraction of the total protein present or when we are dealing with the presence of other membrane constituents, a radioactive concentration marker can be extremely useful. Free amino groups (lysine e-NHz and N-terminal NH2) on the protein can be converted to their N-methyl or N,N-dimethyl derivatives by reductive methylation with [14C]formaldehyde and a reducing agent such as NaBH4 7 or NaCNBH3.8 The protein concentration in each sample can then be established by comparison with the 14C activity of protein standards of known concentration. The tritium content and protein concentration of samples at each time point are determined by 14C-3H dual isotope counting with a two-channel counting technique because the two isotopes have different E-emission energy spectra. However, the energy spectra overlap, so it is necessary to determine the counting efficiency of the two isotopes in each channel to correct for this "spillover. ''9 The 14C-labeled protein acting as a tracer is, as a rule, present in such low quantities that the hydrogen exchange from 14C-modified molecules can be neglected. However, it is important that the ~4C-modified molecules distribute themselves in a heterogeneous system of particles similarly to the native molecules. This is most important in case separation techniques, such as filtration, are used. The validity of nrem values obtained from complex systems 7 G. E. Means and R. E. Feeney, Biochemistry 7, 2191 (1968). s W. Jentoft and D. G. Dearborn, J. Biol. Chem. 254, 4359 (1979). 9 D. L. Horrocks, "Applications of Liquid Scintillation Counting." Academic Press, New York, 1974.

636

PROTONS AND MEMBRANE FUNCTIONS

[47]

by the double-label method can and should be verified by straightforward controls. The sample can be divided, one-half for radioactive counting, and the other for protein analysis such as high-performance liquid chromatography (HPLC) for native proteins or SDS-PAGE gels for more complex systems. The quantitation of Coomassie stain is, of course, not at all precise when compared to 14C label, but multiple determinations at a single time point give a good indication whether the n r e m calculated from double label represents a systematic under- or overestimate. The relative changes of n r e m with time are, of course, little influenced by systematic errors of this type. The isotope enrichment factor discussed previously represents an error of a similar nature. Filtration Methods. When the system of proteins to be investigated consists of large particles, such as red cell ghosts, straight filtration is an appropriate method for moving excess tritium at the first time period and for the removal of lost tritium during the time course of the out-exchange. It is rapid and avoids trapping of liquid, a phenomenon encountered in rapid sedimentation. Using this method, it is often advisable to follow the first filtration by rapid wash steps to eliminate tritium-containing solvent on the filter. It is useful to remove the deposit from the filter in the first filtration step; subsequent filtration steps, as a rule, result in filters that can be counted directly if the protein concentration can be determined by dual-label methods. If the counting efficiency is too low, one can backexchange tritium into buffer by incubating the filters at high temperature. It is advisable to use glass fiber filters of minimal dimeter and always to insert the filter into the counting vial in a similar manner. If one deals with polymer solutions where the polymer states are assumed to be in equilibrium with monomers, then monomers may be lost in filtration. On such occasions, there is a more sophisticated variation of the filtration technique, based on the use of ion-exchange filters, l0 Binding of protein or peptide to phosphocellulose combines some of the techniques of freezing-lyophilization with some of the advantages of other techniques of a second separation. Briefly, the sample is placed on phosphocellulose paper at 0°, pH 3, conditions which minimize but do not abolish further exchange of buffer. The sample is vacuum filtered with several rinses of buffer. The paper is transferred to a concentrated salt solution, which elutes the material from the paper. Then follows radiometric determination of tritium content and determination of protein concentration in the eluate. The time point is uncertain by about 0.5-2 min, the same error as in gel filtration. If the material cannot be stripped from the paper, the filter can be counted directly if 14C is used for concentration markers. ~0A. A. Schreier, Anal. Biochem. 83, 178 (1977).

[47]

HYDROGEN EXCHANGE KINETICS

637

If the precipitate is tightly fixed to the filter and does not loosen in scintillation liquid, the filters can be removed from scintillation vials rinsed with toluene (in a well-ventilated hood), and the dried protein can now be determined by any classical method, such as total nitrogen determination. Dialysis Method. For 3H experiments, rapid dialysis 1~ provides its own separation. The in-exchange solution is transferred, once isotope equilibrium is reached, into a dialysis bag that is stretched over a plastic rack so as to form a thin bilayer sandwich with the distance between the dialysis tubing walls well below 1 mm. For example, a 1-in. wide tubing is stretched to a length of - 1 6 in. Sample size in this case is - 0 . 5 ml. The plastic rack is placed in a cylinder containing 750 ml of buffer and attached to any device providing rapid rotation for stirring. Next, one simultaneously takes samples of the bag contents and of the surrounding dialysate, subsequently subtracting the radioactivity of the latter from that of the former to correct for counts arising from labeled solvent molecules. The method involves no undetermined dilution of material. A large number of parallel experiments may be conducted. Errors creep in if isotope equilibrium across the membrane has not been reached or if background levels of radioactivity approach those of the bag contents. The weak point of the method is the dialysis of the very high activity inexchange solution that provides a high background. It is often advisable for particulate matter to spin the in-exchange solution down to remove the excess solvent and resuspend the precipitate in an equal volume of new buffer before transferring to the dialysis bag. If this is not possible we can discard the dialysate after the first point. This considerably reduces the background for the majority of points. If the system one wants to investigate cannot be stirred, such as in the case of the study of actin-spectrin gels in vitro, the experiments can still be carried out by using special cells where the volume change due to osmotic gradients is kept to a minimum. I will describe the method in detail because the individual steps are pertinent to all rapid dialysis methods, a method suitable for studies of protein polymers and more complex structures. A thin film dialysis cell is shown in Fig. 1. Dialysis membranes were segments of Fisher cellophane tubing 10 mm wide (flat) by 15 mm in length which had been boiled in several changes of distilled water, drained of excess water, and brought to equilibrium at room temperature with water vapor in a sealed container. The dialysis buffer contained polyethylene glycol (Baker) with a molecular weight of 20,000 in equal molality to the sample to prevent dilution of the solution during dialysis. The cell was H S. W. Englander and D. Crowe, Anal. Biochem. 12, 579 (1965).

638

PROTONS

AND

MEMBRANE

[47]

FUNCTIONS

o

©

]gg r~

I

/ --

....

""

~"-,

© I

I

I II

I

LX'. . . . .

it .,7 . I~------'.'J

.

I .

.

.

i /

©

© o

~

2

I

' ~

b,\\",~

,

FIG. 1. Dialysis cell consisting of two similar plates machined from Lucite. They are clamped together with a Teflon gasket between them by four screws. Inlet and outlet tubes are 17-gauge stainless-steel needle tubing cemented in place. The segment of dialysis tubing is shown as dotted lines. The dimensions are given in inches. From P. Hallaway and B. E. Hallaway, Arch. Biochem. Biophys. 234, 552 (1984) with permission.

loaded by clamping one end of a segment of dialysis tubing with a clamp and adding 15/zl of in-exchange solution through the other end. This end was also clamped to close the tubing and then both clamps were removed and the tubing clamped by two sides of the cell. After flushing briefly with nitrogen, the cell was filled with dialysis buffer, sealed, and placed in a thermostated box at 25 °. Any number of cells can be prepared at one time. They were connected in parallel to the dialysis buffer supply to begin outexchange. Flow rate, controlled by a pump, was 9.5 ml/min for the first 15 min and gradually decreased over the next 7 hr to 1 ml/min. At various times, up to 15,800 min, a cell was removed for assay. After draining out the dialysate, the segment of tubing was clamped at one end and the other end was trimmed. The gel was removed with microspatula and the spatula with adhering gel placed in 2 ml of buffer ice bath with occasional agitation until dissolved. A 50-/zl aliquot was counted in 10 ml of toluene counting fluid containing 10% BBS-3 (Beckman) as solubilizer. The remainder of the solution was used to determine the protein concentration. On occasions, when the gel does not dissolve, it can be weighed and counted directly. The protein concentration, in this case, was determined

[47]

HYDROGEN EXCHANGE KINETICS i

i

i

i

1.8

i

639 T

i

i

i

B

A

1.4

--

1,2

08 log

I (rain)

i

c

12

D

f,\ x \ x \

tO

\ \x

8

/U% y

6

4

2 t 0 0

-f

-2

,

-3

.~

-5

-;

0

log k

-i Crnin

-2

"5

-4,

-5

-6

tl

FIG. 2. (A) Comparison of hydrogen exchange cyanomethemoglobin A by gel filtration (O ) and microdialysis (0---) in phosphate buffer at pH 7.0, ionic strength 0.2, and 25°. H(t) is the number of hydrogens per Hb chain remaining unexchanged at time t. (B) Same studies for deoxyhemoglobins in gel by microdialysis (0) and deoxyhemoglobin A in solution by gel filtration (O). (C, D) The probability density distribution functions of rate constants derived from data in (A) and (B). From P. Hallaway and B. E. Hallaway, Arch. Biochem. Biophys. 234, 552 (1984) with permission.

by dual label or by standard protein assays such as nitrogen determination on another weighed sample of the gel. The effectiveness of the dialysis cell was determined by comparing the exchange from a concentrated hemoglobin solution in the cell and in a parallel experiment using the same protein concentration, but with Sephadex columns for separation. The data are shown in Fig. 2. The agreement for data points above 10 min is excellent. The use of rapid filtration techniques for the first step and double label allows measuring times somewhat below 10 min. However, it is not reasonable to expect reliable data for time points below 5 min.

640

PROTONS AND MEMBRANE FUNCTIONS

[47]

Deuterium Exchange by Infrared Measurements When deuterium isotope replaces hydrogen on the nitrogen of the peptide bond, the amide II frequency at 1552 cm -~ is shifted by 100 cm -1 , whereas amide I frequency, dominated by carboxyl motion, remains essentially unchanged. Consequently, changes in the ratio of amide II/amide I describe the time course of deuterium replacing hydrogen on the peptide nitrogen. The method is specific for the peptide bond. Side-chain hydrogens do not contribute to the observed changes. The fraction of exchange at each time point is expressed by

x(t)

=

An (t) - A I I (2) toAi

(6)

where AII(t) is the intensity of the amide II band at time t, All(to) is the intensity of the amide II band of the fully deuterated protein, A1 is the intensity of the amide I band, and to the ratio Au(O)/AI(O) for the fully hydrated protein in 2H20. The intensities of the amide I and lI bands can be estimated from the peak heights or peak areas. AI is measured with reference to a baseline drawn between the absorption minima on either side of the amide I band while An(t) may be estimated with AII(~) as the baseline. A determination of to cannot be made directly and its value is generally established by extrapolating the intensity changes to zero time under conditions that minimize exchange. Values of to lie between 0.4 and 0.5 for proteins. Further details of the methods can be found in Ref. I. Since H20 absorbs strongly in the wavelength region of interest, dilution of small volumes of aqueous protein solution into 2H20 is not possible, and exchange is initiated instead by dissolving dry protein in appropriately buffered 2H20. Exchange of proteins that are sensitive to freezedrying can be initiated by passage of the protein solution through a Sephadex G-25 column previously equilibrated with 2H20. The solution of protein in ZH20 is then rapidly transferred to a thin cell (i.e., 0.1 mm) with CaF2 windows and the sample spectrum between 1700 and 1400 cm -j recorded as a function of time. Infrared methods are not sensitive to light scattering and even very opaque solutions can be studied. However, one must note two things. If it is suspected that the suspension is not stable and sedimentation takes place, then the cell must be inverted regularly to keep the variation in density down. A second point is the fact that CaF~ windows are not suitable for systems sensitive to Ca 2+ levels. As an example of the suc-

[47]

HYDROGEN EXCHANGE KINETICS

641

cessful use of the method, we offer that for the study of sarcoplasmic reticulum vesicles. ~z The purified vesicles are dialyzed repeatedly in buffer of desired pH and salt composition. The protein concentration is adjusted to give a final value between 30 and 18 mg protein/m]. The suspension is divided into 0.1-ml aliquots and the aliquots are freeze-dried. The exchange is initiated be dissolving an aliquot in 60-80/zl of 2H20 (final protein concentration should vary between 3 and 8%). The suspension is transferred to a cell of 50/zM thickness. The baseline and the ratio, AII/AI, for total in-exchange was determined by 1.5-hr exchange at 60°. The initial peak ratio was assumed to be 0.45. At this point, it is important to remember the difference between pH and p2H: p2H = pH + 0.4, where pH represents the reading of the pH meter.

Comments on the Mechanism of Hydrogen Exchange If the apparent rate constant of the exchange observed, kapp, c a n be written as flkex, then in order to determine a value for/3, the conformational contribution, one has to know the properties of kex for both the peptide group and the exchangeable side-chain configurations. Extensive studies have been carried out on model peptides and polypeptides. 1,2,13It is known that the chemical exchange rate constant k~x for any site is a sum of the contributions by hydroxyl ion, hydrogen ion, and water catalysis: k~x = k0 + kH(H) + koH(OH)

(7)

As expected, the individual constants kn and koH are changing with the primary structure in the immediate vicinity of the exchanging site, ionic strength, pressure, and temperature. The pertinent literature has been reviewed repeatedly.l.Z In addition, attention should be given to a recent report about N versus O protonation during the exchange reaction.~4 The question pertinent to the studies in more complex systems is the legality of substituting kex values determined for peptides into the expression for kapp. The variation of the rates due to the variation in the primary structure of the peptide chain, although quite pronounced, is not overwhelming when compared to the very large variation of the conformational factor/3; thus, instead of constructing a distribution of k~× values for the polypep12 y . Kirino, K. Anzai, H. Shimizu, S. Ohta, M. Nakanishi, and M. Tsuboi, J. Biochem. 82, 1181 (1977). 13 A. Ikegami, M. I. Kanehisa, M. Nakanishi, and M. Tsuboi, Adv. Biophys. 6, 1 (1974). 14 B. D. Tuchsen and C. K. Woodward, J. Mol. Biol. 185, 421 0985).

642

PROTONS AND MEMBRANE FUNCTIONS

[47]

tide chain from available data for peptides, 15a simplified form for average (kex), based on the constants for poly(DL-alanine), has been introduced by the Carlsberg laboratories. 16 (kex) = 5 0 ( 1 0 -pH + 10 pH-6) × 1010"05(t°-20~1

(8)

The justification for substituting such an estimated kex value into kapp rests on the assumption that the attenuation of the exchange rates observed in protein structures is due to the shielding of the sites from water, but also that there is a certain probability of finding the site in an unfolded state, and, most important, that the conditions for exchange from such an unfolded or accessible state are comparable to those encountered in the studies of dilute solutions of poly(DL-alanine). The simplest structural model describing this is a simple equilibrium for partial or full unfolding with no exchange taking place from the native state. The issue of whether hydrogen exchange takes place by such a simple system of thermal unfolding reactions or whether the structural fluctuations in the native state are responsible for providing pathways of exchange is hotly debated, the discussion of which ~-3 is out of place in this context. However, it is important to remember that the situation for protein complexes and particles of higher order is probably more complex in terms of defining a simple unfolding mechanism responsible for exchange. The issue is important if one wants to compare the properties of an isolated protein in dilute solution and the properties after the protein is inserted into a membrane bilayer or participates in a cytoskeletal complex. For example, a change in the relative efficiency of hydroxyl ion and direct water catalysis has been proposed for conditions far from the dilute state. 17 For study of the effects of changes in temperature, ligation, or physical state, a type of investigation many hydrogen exchange studies are concerned with, one measures relative changes or rates due to changes in conditions, and the above problem is of lesser importance. Another note of caution: When calculating activation energies I for the exchange, the contribution due to kex can vary considerably due to the difference between the activation energies for the ion-catalyzed exchange, and the presence of enthalpy represents the temperature dependence of Kw. Temperature studies at constant pH and at constant pOH present different enthalpy values. 15 R. S. Molday, S. W. Englander, and R. G. Kallen, Biochemistry 11, 150 (1972). ~ A. Hvidt and S. O. Nielsen, Adv. Protein Chem. 21, 287 (1966). 17 R. B. Gregory, L. Crabo, A. J. Percy, and A. Rosenberg. Biochemistry 22, 910 (1983).

[47]

HYDROGEN EXCHANGE KINETICS

643

Studies of Model Systems The intention in presenting the following section is to provide the reader with an understanding of what kind of model systems are pertinent to the efforts of understanding the exchange kinetics of membrane-associated systems.

Hydrogen Exchange Kinetics of Proteins in Crystalline State Although studies of exchange from protein crystals have been few, careful studies have established that there is very little, if any, difference between exchange from'crystal and solution, 18.19 and this allows us to draw some very tentative conclusions about the changes to expect when proteins line up in crystalline arrays. First, the high concentration, certainly producing very nonideal conditions 2° and admittedly changing the activity of water, has little effect on the rates of exchange. The experiments are usually carried out at constant pH, and such behavior is to be expected if the presence of protein does not influence Kw seriously. It also tells us that the hydration sheet surrounding the protein molecule is in equilibrium with bulk solvent and that equilibrium is not appreciably challenged when molecules line up in crystalline arrays. These observations and conclusions can be stretched somewhat more based on the results of a study of the exchange of protein powder or crystals without mother liquid. The experiments were carried out as a function of vapor pressure which allows variation of the extent of hydration. zl Although a comparison of the exchange at such conditions, and exchange in solution is quite difficult, and the results are somewhat ambiguous due to difficulties in defining ionic strength, one point becomes quite clear. Starting with dehydrated protein, the exchange rate increases substantially with increasing hydration up to 0.15 g water/g protein, a situation where only about one-half of the full hydration sheet (0.38 g/g) is present. Above this level, the exchange is no longer sensitive to vapor pressure increases, although other physical properties, such as the specific heat capacity, have not yet leveled off. It must be concluded that the water of hydration is extremely mobile and that the exchange is of a low order in water concentration. Large cooperative hydration processes are not necessary to achieve exchange. ~s E. Tuchsen, A. Hvidt, and M. Ottesen, Biochimie 62, 563 (1980). 19 G. A. Bentley, M. Delepierre, C. M. Dobson, R. E. Wedin, S. A. Mason, and F. M. Poulsen, J. Mol. Biol. 170, 243 (1983). 2o p. D. Ross and A. P. Minton, J. Mol. Biol. 112, 437 (1977). 21 j. E. Schinkel, N. W. Downer, and J. A. Rupley, Biochemistry 24, 352 (1985).

644

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These observations and the near normal exchange from cross-linked crystals in mother liquid Is have been interpreted as supporting an exchange mechanism described by small-amplitude noncooperative fluctuations and not partial unfolding reactions. Nonetheless, the importance of these investigations is to establish a relationship between the degree of hydration and exchange properties, a question of great importance in studies of membrane-associated proteins. The effectiveness of small amounts of water in accomplishing the exchange would point to bond breaking and structural rearrangement as the slow step that rapidly becomes rate limiting. This consideration leads directly to the consideration of solvent viscosity.

Solvent Viscosity and Exchange Hydrogen exchange from proteins shows strong dependence on viscosity, z2 The experiments are difficult because additives such as glycerol very clearly change the activity of hydrogen and hydroxyl ions. The faster exchanging hydrogens show a linear dependence on viscosity, whereas the very slow ones show a more complex dependence. The latter is not surprising because we are dealing here with both effects on stability and on kinetics. It is assumed that the fluctuations leading to exchange from the slowest hydrogens involve large amplitude fluctuations best described as chain melting or unfolding. It is interesting to note that the fluctuations leading to fluorescence quenching by the acrylamide, another method for study of protein dynamics, 23 show the opposite effect. It is the slowest hydrogen, in this case, that shows no viscosity dependence. This would indicate that the rate-determining steps are different, although considering the very rapid nature of the quenching reaction, peptide bond breakage should even in this case be the rate-limiting step. If one combines this with the observation that the attenuation of the fluorescence quenching rates covers only two orders of magnitude compared to the eight magnitudes seen in hydrogen exchange, one might speculate that the two methods are seeing different aspects of the processes involved in structural dynamics.

Effects of Protein-Protein Association and Polymerization Protein association reactions present an interesting picture. First, strong noncovalent complex formation such as the tetramer-dimer equilibrium of hemoglobin leads to clear-cut attenuation of the exchange rate 22 R. B. Gregory and A. Rosenberg, in "Biophysics of Water" (F. Franks and S. Mathias, eds.), p. 238. Wiley, New York, 1982. 23 M. R. Eftink and C. A. Ghiron, Anal. Biochem. 114, 199 (1981).

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of the tetrameric form. The changes are large enough that the difference can be used to determine the equilibrium constant of the association reaction. 24 The extent and nature of the changes associated with proteinprotein association have been most thoroughly studied in the case of trypsin reacting with bovine pancreative trypsin inhibitor. 25 It is evident that in this tight bond the binding free energy is distributed, leading to strengthening of hydrogen bonds and restrictions of segmental motion. The bulk of the effect is localized in or near the interface, but some fraction of the energy propagates quite far into the polypeptide matrix of both proteins. When one compares the result of these studies with observations of protein polymers, a surprising difference appears. A very thorough study of deoxyhemoglobin fibers, multistranded helical structures, showed no appreciable change in the exchange rates when hemoglobin tetramers assembled to long fibers, z6 These findings seem not to be unique. Unpublished studies from this laboratory have established that G-actin monomers show very marginal changes in their hydrogen exchange properties when assembled into F-actin polymers. This is quite remarkable because changes, mostly attenuation, of rates for protein can be observed even with binding of quite small molecules, such as coenzymes and inhibitors. There are several possible reasons for this dichotomy to appear. First, one can distinguish between a strong bond between two molecules of a dimer with considerable free energy per interface and a three-dimensional assembly, similar to the crystal phase, where the strength of the structure may be due to relatively weak cooperative interactions between multiple individual protein molecules. The free energy of interaction for the individual interface may be associated with modest energies in the last case. Another more mechanistic explanation could be based on the question whether the individual sheet of hydration remains intact after association or whether the binding reaction leads to release of bound water. Polymers assembled utilizing crystal-type weak forces would be expected to retain their original complete sheet of hydration. At this point, a review of the kinetic aspects of the problem of protein association is in order. Consider a single exchanging site on a monomer exchanging with the rate constant k~ and the same site in the polymeric state exchanging with a rate constant k2. Then, at any arbitrary degree of polymerization, the observed rate constant can be written as kapp -- klt~ +

(1 - ~b)k2

24 A. D. Barksdale, and A. Rosenberg, this series, Vol. 48, p. 321. 25 C. K. Woodward, J. Mol. Biol. 111, 509 (1977). 26 B. E. Hallaway and P. E. Hallaway, Arch. Biochem. Biophys. 234, 552 (1984).

(9)

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where ~b stands for the fraction of time the site is in the monomeric state. 2z Consequently, in case the site in the polymeric state does not exchange at all and we have a time-averaged monomeric concentration of 10%, one sees an apparent rate attenuation by a factor of I0, underestimating the change associated with the polymeric state. The form of the kinetics observed depends very much on the relative rates of the association reaction and the exchange. The wide spread of exchange rates allows us to choose rates slow enough so that the association reaction can be considered as fast, which simplifies the kinetic model for exchange. 27 The final comment concerning the polymer state is the question of separate status for the end molecules of a polymer chain. The question is quite similar to that for long helical polymers where the difference between for residues according to their position has been discussed thoroughly. 28

Protein Surface Interactions One can consider absorption of a protein molecule on a surface as an asymmetric dimer formation where one reactant, the surface, has properties vastly different from protein molecules. The resulting changes in hydrogen exchange kinetics of proteins have been studied in the case of the absorption of myoglobin and hemoglobin on silica particles. 29 The most interesting observation was that the exchange rates of monomeric myoglobin were attenuated, as expected for strong noncovalent binding on a hydrophobic surface. However, the hemoglobin tetramer, when bound, showed opposite behavior. There are two possible explanations. First, binding may result in a shift of the tetramer-dimer equilibrium and since the dimer exchange is faster, an apparent enhancement of rate may be observed. The attenuation expected for binding reaction would consequently be obscured. As a second alternative, the observed change can be due to the strain introduced in the tetrametric molecule when it is bound strongly by one of its dimeric subunits. In order to accommodate the optimal interactions with the surface, changes in the dimer interface may result in loosening of the structure at some other location. The resulting rate enhancement compensates for the attenuation observed at the interface between the protein and silica bead. 27 p. E. Hallaway, B. E. HaUaway, and A. Rosenberg, Biochemistry 23, 266 (1984). 2s W. G. Miller, Biochemistry 9, 4921 (1970). 29 B. E. Hallaway, P. E. Hallaway, W. A. Tisel, and A. Rosenberg, Biochem. Biophys. Res. Cornrnun. 86, 689 (1979).

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Examples of the Use of Hydrogen Exchange Kinetics in the Study of Membrane Systems The two systems that have attracted the most interest are rhodopsin and sarcoplasmic reticulum. A respectable number of studies of rhodopsin and bacteriorhodopsin have been published. 3°-34 Despite the original underestimate of slowly exchanging hydrogens which turned out to be slow enough not to exchange at all, it has become clear that rhodopsin has an extremely stable and solvent inaccessible core. This is unusual to the extent that we see even a number of side-chain protons exchanging quite slowly. In smaller globular proteins, this is not the case. Hydrogen exchange kinetics and IR specra combined have led to the proposal that the core consists of closely packed helical segments. It has been shown that the exchange properties of membrane-bound and detergent-solubilized rhodopsin are quite similar except for their very different sensitivity to bleaching. It becomes clear that besides identifying a core and conditions that permit the core to exist, hydrogen exchange kinetics based on interpreting out-exchange curves in a comparative fashion do not yield further information. The more sophisticated exchange methodologies developed recently should preferably be used in future studies. The second membrane system that has been studied systematically is the sarcoplasmic reticulum. ~2 The studies here have had the advantage that the preparation evidently can be freeze-dried, allowing an advantageous use of the IR method. This is in contrast to the work with rhodopsin described above that did not allow study of fast hydrogens by infrared because the change into the exchanging buffer had to take place by dialysis. The data handling encountered in the investigations of sarcoplasmic reticulum has been somewhat more sophisticated, with the use of qualitative distribution functions, the so-called relaxation spectra. 2°,35-37The investigators also make use of the temperature dependence of the exchange process. Their approach is to correlate the Arrhenius plots with similar plots from studies of other membrane properties. They show that the well-known temperature-dependent membrane transitions seem to influence only one fraction of the exchanging sites. The temperature depen3o N. 3~ H. 32 H. 33 H. 34 T. 35 K. 36 K. 37 K.

W. Downer and S. W. Englander, J. Biol. Chem. 252, 8092 (1977). B. Osborne, FEBS Lett. 67, 23 (1976). B. Osborne, and E. Nabedryk-Viala, FEBS Lett. 84, 217 (1977). B. Osborne, and E. Nabedryk-Viala, Eur. J. Biochem. 89, 81 (1978). Konishi and L. Packer, FEBS Lett. 80, 455 (1977). Anzai Y. Kirino, and H. Shimiza, J. Biochem. 84, 815 (1978). Anzai, Y. Kirino, and H. Shimizu, J. Biochem. 90, 349 (1981). I. Higashi and Y. Kirino, J. Biochem. 94, 1769 (1983).

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dence of the faster hydrogen shows a break at the temperature of the observed membrane transition. This leads to the conclusion that whereas the surface hydrogens directly see charges in the membrane bilayer, the core hydrogens remain uninfluenced. In an exemplary way, the hydrogen exchange results are correlated with other independent methods, in this case the spin-label method. In conclusion, one can summarize the type of information to be extracted from hydrogen exchange kinetics for the study of membranes. (1) The question of how the rates for the exchanging sites reflect the existence and relative size of a stable structural core can be answered by construction of distribution functions from the data gathered over a wide period of time and pH range. (2) Where in the molecule are these structures located? This is a more difficult question which for small molecules can be tackled by NMR and neutron diffraction. In membrane proteins, the method of choice would be the proteolytic cleavage of proteins 38 and the study of the behavior of their fragments. (3) What kind of motion do the different segments of the protein undergo? Here studies of pH, temperature, and viscosity dependence provide the best avenue of approach. (4) What is the time range of the motions? Here hydrogen exchange should be correlated with methods such as fluorescence quenching and spin label. (5) What is the biological relevance of the motion? This can only be established if one can correlate the presence of patterns of exchange, such as very tight cores, with activity. Studies of families of proteins with similar activities seem at present to represent the most promising approach.

Acknowledgment This work was supported by NSF PCM 800 3744.

38 j. j. Rosa and F. M. Richards, J. Mol. Biol. 133, 399 (1979).