Investigation of higher order structures of proteins by ultraviolet resonance Raman spectroscopy

Prog. Biophys. molec. Biol., Vol. 58, pp. 1-18, 1992. Printed in Great Britain. All rights reserved.

0079-6107/92 $15.00 © 1992 Pergamon Press Ltd

INVESTIGATION OF HIGHER ORDER STRUCTURES OF PROTEINS BY ULTRAVIOLET RESONANCE RAMAN SPECTROSCOPY TEIZO KITAGAWA Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki 444, Japan

Abstract--Progress in laser technologyand light detection deviceshave enabled us to exploreprotein structures and their dynamics by using time-resolvedresonance Raman spectroscopy.It is in the last decade that Raman spectra of proteins excited at 200-240 nm have brought about rich structural information. The technological developments in deep UV resonance Raman spectroscopy are reviewed first, and the unique information on proteins obtainable from such spectra are summarized. As an application of this technique to investigationsof the higher order structures of proteins, studies on the quaternary structure transition of haemoglobin are described. CONTENTS I. INTRODUCTION

II. EXPERIMENTALSECTION 1. Source 2. Sample Illuminating System 3. Detection System

III. UV RR SPECTRA 1. 2. 3. 4. 5. 6.

Amino Acid Solutions Secondary Structure of Polypeptide Cis-amide Indicator and Conformation Sensitive Band Imide Vibration of Proline Markers of Local Environments Discernment of Higher Order Structures

IV. QUATERNARY STRUCTURES AND ITS DYNAMICS IN HAEMOGLOmN

1. 2. 3. 4.

Quaternary Structure Marker Continuous Strain Model Time-Resolved UV RR Spectra Bohr Effect and a Mechanism of Quaternary Structure Change

7 7 9 10 14 17 17

ACKNOWLEDGEMENTS REFERENCES

I. I N T R O D U C T I O N Higher order structures of proteins, including a folding mode of a polypeptide chain (secondary structure), the relative arrangements of the folded segments (tertiary structure) and organization of protomers (quaternary structure), play essential roles in the regulation of activity of various proteins. A typical example is an allosteric effect, in which binding of a ligand such as a substrate or product of enzymic reactions to a regulation site of the enzyme, causes an activity change. The activity regulation is achieved through a conformation change of the protein, but little is known about detailed structural mechanisms of the allosteric effect. X-ray crystallography has provided a basis for studying the structure-function relationships of proteins. This method provides a three-dimensional structure of a whole molecule. It often occurs, however, that a limited part of a large molecule plays a key role in its functioning and one is curious to know the active part in more detail. Such a demand is not generally fulfilled by the X-ray picture of a molecule, particularly for large proteins. The X-ray analysis may be likened to looking at a complicated large molecular model in the dim light. In contrast, various spectroscopic techniques m a y be likened to illuminating a limited part of the molecular model with a small but bright flashlight. If the illuminated part is an active site, the observation would bring about substantial information on this molecule. ' V i b r a t i o n a l spectra of molecules are sensitive to inter- as well as intra-moleeular

2

T. KITAGAWA

interactions, and have been extensively used in structural analysis. Among several methods for measuring the vibrational spectra, resonance Raman (RR) spectroscopy has attracted the attention of biochemists over the last few decades (Carey, 1982; Tu, 1982; Clark and Hester, 1986; Spiro, 1987). This is a technique to observe the vibrational spectrum ofa chromophore selectively by tuning the wavelength of Raman-exciting laser light to an absorption band of the molecule. Since an RR spectrum can be measured with a small amount (10-100 ~tl) of a dilute solution (10- 3-10 - 6 M), it has been extensively applied to chromophoric proteins with visible absorption. Owing to developments of pulse lasers and array detectors, time-resolved measurements on a nsec time scale are now becoming daily events in Raman laboratories. Experiments using the 363.8 nm line of an Ar ÷ ion laser (Brown et al., 1977) or the second harmonic of its 514.5 nm line (Sugawara et al., 1978), suggest the potentiality of UV RR spectroscopy. Recently it was demonstrated that, when the wavelength of Raman excitation light is tuned to 200-240 nm, the intensity of some Raman bands of aromatic residues are strongly enhanced (Rava and Spiro, 1984, 1985a; Johnson et al., 1984; Mayne and Hudson, 1987). The intensity enhancement of Raman bands in the UV excitation is anticipated from the absorption spectra of some aromatic amino acids shown in Fig. I. Since the time resolution of this technique (10-12 sec) is much faster than that in NM R (10- 6 s e e ) , which is currently the most common technique for studying structure of proteins in solutions, UV RR spectra combined with time-resolved measurements are expected to bring about the dynamic features of protein conformation changes.

/~, II /I \ i, / /~ i i / ~i

I

Tyroslne

'

Tyroslnote

Tryptophan Phenylalanlne

LI / I l

200

~ CO

,

e-

e-

I

t~

tD

240

280

Wavelength (nm)

FIG. 1. The UV absorption spectra of aqueous solutionsof aromaticaminoacids.Tyrosinateis the 0.1 ~t NaOH solutionof tyrosine. Accordingly, in this paper, recent experimental developments of UV RR spectroscopy are summarized first, and then applications to the study of protein conformations are reviewed. Finally, the current situation of the UV RR spectral studies on the quaternary structure of haemoglobin (Hb) is explained in detail, since Hb has served as a typical model protein of general allostery (Monod et al., 1965), and rich information on it has offered a fertile ground for scrutinizing various ideas about the mechanism of cooperative oxygen binding (Shulman et al., 1975; Perutz, 1979). II. EXPERIMENTAL SECTION 1. Source A breakthrough for UV RR spectroscopy was made by Ziegler and Hudson (1981, 1983), who succeeded in using the fifth harmonic (212.8 nm) of a Nd:YAG laser as a source of Raman scattering of benzene. Slightly later, Asher et al. (1983) generated UV light around

Higher order structures of proteins

3

220-250 nm by mixing the dye laser output excited by the second harmonic ofa Nd :YAG laser with its fundamental, and applied it to measurements of UV RR spectra of proteins (Dudik et al., 1985a; Johnson et al., 1986; Asher et al., 1986; Johnson and Asher, 1987). Rava and Spiro (1984, 1985a) obtained the 218 and 200 nm lines using stimulated Raman of H2 (7 atm.) excited by the fourth harmonic of a Nd :YAG laser, and applied them to observe the UV RR spectra of Trp and Tyr successfully. Spiro and coworkers (Caswell and Spiro, 1986, 1987; Copeland et al., 1985; Copeland and Spiro, 1985, 1987, 1986a; Fodor et al., 1986; Hildebrandt et al., 1988; Rava and Spiro, 1985b; Sorrel et al., 1986b) obtained the UV RR spectra of various proteins using the H2 Raman-shift method which gave the shortest source at 184 nm. Hudson and coworkers developed the stimulated Raman technique to generate the shortest excitation source at 141 nm and applied it to measure RR spectra of benzene (Gerrity et ai., 1986), ethylene (Sension et al., 1987), and amino acids (Mayne and Hudson, 1987). These UV sources based on a Nd:YAG laser give rise to 10 nsec pulses with 10-30 Hz repetitions and yield a relatively high peak power. Accordingly, appreciable numbers of molecules are excited to a triplet state by the Raman probe pulses. If relaxation of the excited molecules were slower than the period between two successive laser pulses, the Raman intensity would become weaker than expected due to depletion of molecules in the ground state. In fact, when the flux photon density is high, Raman intensity is not proportional to the intensity of the excitation light and exhibits saturation (Teraoka et al., 1990; Harmon et al., 1990; Suet al., 1990). Asher and coworkers (1987) recommended the use of the second harmonic of the high repetition rate (~ 200 Hz) excimer laser-excited dye laser output for the 220-260 nm region, although there is no good harmonic generator for the wavelength region shorter than 220 nm. Recently, Rodgers et al. (1992) obtained beautiful UV RR spectra of Hb using this method. 2. Sample Illuminating System

A matter of concern in UV RR spectroscopy is damage to the sample due to illumination in strong laser light. To circumvent this problem, a spinning cell is usually used in the visible excitation, but in the UV excitation the scattering from the cell wall often seriously raises the background level of Raman spectra. The more popular way to make UV RR measurements is to flow a sample, although this method requires a relatively large amount of sample. An example of the sample illuminating system with a back-scattering geometry for the flowing sample without glass capillary is illustrated in Fig. 2(A), where the excitation light is made into a rectangular shape similar to the image of the entrance slit of the monochromator by passing it through two cylindrical lenses. It is necessary to reduce the flux photon density of the excitation light without reducing the total number of incident photons.

(A)

FI Lens

(B)

from PerlsloPump o.s÷

Sample jet

"~

~

_.~

" Prlsm

mple Stream Shee!

Cylindrical lens

/~ Long focul lens / /.~ Prism

to Reservoir

Sample Flow System(SSS)

FIG. 2. Sample illuminating optics (A) for UV Raman experiments and the wire-guided flowing device (B),

4

T. KITAGAWA

For solutions containing amino acids and small proteins, the slow stream spouting from the capillary (diameter=0.8 mm) as shown in Fig. 2(A) is sufficient, but this is not satisfactory for large proteins, since a foam is occasionally formed, and the surface of stream is disordered, which yields serious noises in a Raman spectrum. In such a case a wire-guided flow system, illustrated in Fig. 2(B) (Kaminaka et al., 1990), gives good results. The solution is flowed between two thin wires which are in touch with the sample reservoir at the bottom, and the distance between the two wires would be automatically varied by the surface tension of the solution. 3. Detection System Since Raman scattered light is very weak, effective collection of scattered light is extremely important. Although the forward transmission geometry using convex lenses is general, the reflection geometry using a concave mirror (Asher et al., 1983), or Cassegrainian collection system (Takeuchi and Harada, 1990), is used to raise the F number. As a detector, a solar blind UV photomultiplier (Hamamatsu, R 166UH or R955) (Rava and Spiro, 1984; Ziegler and Hudson, 1983; Kaminaka et al., 1990) or diode array (PAR 1420B, PI, D/SIDA-700/G) (Asher et al., 1983; Suet al., 1990; Takeuchi and Harada, 1990) is used in practice. In the latter case, removal of stray light is a prerequisite. Accordingly, a large double monochromator (Teraoka et al., 1990; Ames et al., 1990) or a triple-monochromator (Spex 1877) (Asher et al., 1983) is used. The spectra shown in this paper were obtained (Kaminaka et al., 1990) by using a large single-monochromator (Spex 1269) with the solar blind photomultiplier (Hamamatsu R166UH), for which the holographic grating (2400 grooves/ mm) was used in the second order.

III. UV RR SPECTRA 1. Amino Acid Solutions Figure 3 displays the UV RR spectra of tyrosine (Tyr, A), tyrosinate (Tyr-, B), phenylalanine (Phe, C) and tryptophan (Trp, D) excited at 239 (upper) and 218 nm (lower). The samples are 1 mM aqueous solutions of a given amino acid containing 0.2 M NaCIO 4 in common, which gives a Raman band at 932 cm- 1. Since the intensity of the 932 cm- 1 band of CIO~ is scarcely varied by excitations at different wavelengths around 200-240 nm (Dudik et al., 1985b; Song and Asher, 1991), this band can serve as an internal intensity standard. In accord with the absorption spectra shown in Fig. 1, Raman bands of Tyr- are strongly intensity-enhanced upon excitation at 239 nm, and those of Trp are enhanced at 218 nm. The excitation profiles of aromatic amino acids were measured by Asher et al. (1986) who discussed a relation between the excitation profiles and the properties of the excited electronic state and/or the ground state vibrational modes. Even for a simple amino acid solutions, a radical cation is formed when the laser power is high; Tyr cation radical is reported to give characteristic Raman bands at 1402, 1510 and 1565 cm-1 (Johnson et al., 1986). It must be noted that for measurements of the excitation profile for a compound such as SO~-, SeO 2-, CIO,~, or cacodylate, which is mixed with the sample solution to yield a Raman band serving as an internal intensity standard, may alter the protein conformation (Song and Asher, 1991). One should therefore be careful to choose a suitable compound. 2. Secondary Structure of Polypeptide It is well known that the frequencies of amide modes can distinguish between the secondary structures of a polypeptide (Miyazawa et al., 1958; Krimm and Bandekar, 1986). While the (nrr*) transition of the - C O N H - g r o u p is located around 190 nm, the a-helix and fl-sheet exhibit hypochromism and hyperchromism, respectively. Therefore, for a given excitation wavelength, Raman intensity of the peptide group depends on the secondary structure. Copeland and Spiro (1986b, 1987) determined the Raman cross section of amide II (N-H in-plane bending coupled with the C - N stretching) upon excitation at 192 and 200 nm for proteins whose secondary structure distributions had been determined by other methods,

Higher order structures of proteins

(A)Tyroslne I mM

5

(C)PhenylalanlnetmM

t

o ; ~)

m

(B)

Tyroslne I --" [in O.IM NoOH)

(D) Tryptophon

~

l

I

O4

•, X

I

(C)Phenylalonlne ImM

(B)Tyroslne(In 0.1MNaOH) I~

~--

(D) Tryptophon I mM ,,--

T,~

FIG. 3. UV RR spectra of 1 M aqueous solutions of aromatic amino acids. All samples contain 0.2 M NaCIO 4 which gives rise to a Raman band at 932 c m - ~ as an internal intensity standard. Upper: 239 nm excitation, Lower: 218 nm excitation,

and applied the results to quantify the contents of each secondary structure of new proteins. Song and Asher (1989) used two bands around 1550 and 1390 cm- 1 obtained from a single excitation wavelength for similar quantitative analysis of the secondary structures. Although such analysis could be done with Raman spectra of visible excitation (Lippert et al., 1976), the sensitivity is higher with UV RR spectra. 3. Cis-amide Indicator and Conformation Sensitive Band

It has also been pointed out that the Raman band around ~ 1400 cm- 1 is sensitive to the secondary structure, since the 218 nm excited RR spectrum of polyglutamic acid gave it at 1397 cm-1 for non-regular structure, at 1328 cm-1 for s-helix, and at 1393 and 1445 cmfor E-sheet, and the band was assigned to an overtone of amide V (N-H out-ofplane deformation) (Song et al., 1988). In the same study, a band at 1496 cm -~ of N-methylacetamide (NMA), a model compound for the peptide linkage of proteins, was assigned to the overtone of amide V (Song et al., 1988). However, Wang et al. (1989) noticed that the intensity of the 1496 cm- x band of NMA depended upon laser power after excitation at 200 nm and assigned it to amide II of the photo-isomerized cis NMA. They assigned the band at 1390 cm- ~to the overtone of amide V.of trans NMA and named this band as amide S due to its sensitivity to the secondary structure (Wang et al., 1989). Song et al. (1991) reexamined NMA and admitted that NMA suffers trans to cis photo-isomerization upon

6

T. KITAGAWA

laser illumination around 200-220 nm, and that the cis isomer gives amide II at 1490 cm- 1 with a 10-fold larger cross section than that of the trans form upon excitation at 220 nm. Since the cis amide II involves much less contribution from the N-H in-plane bending mode than the trans amide II, its deuteration shift (1496--* 1480 cm - 1) is much smaller than that of the trans form (1581--,1504 cm-1). On the other hand, Wang et al. (1991a) also reexamined NMA and found that amide S of NMA (1385 cm -1) disappeared upon deuteration of acetyl group, and accordingly reassigned it to the C-H deformation mode. The sensitivity of this band to the secondary structure was interpreted in terms of vibrational coupling with nearby amide III; since the coupling becomes larger in the fl-sheet and loop structures, its intensity is proportional to the non-helical content. Furthermore, Wang et al. (1991b) noted for NMA that the amide I (C--O stretching) frequency is linearly correlated with the solvent acceptor number (H bond donating tendency). The amide I Raman band of NMA is stronger and of higher frequency in non-hydrogen-bonding solvent than in water, while amides II and S are weaker and of lower frequency there. 4. Imide Vibration o f Proline

Proline (Pro) is located at the turning point of the chain in a protein tertiary structure, and the peptide bond of the X-Pro linkage can be cis or trans. Mayne and Hudson (1987) noticed that the UV RR spectra of Gly-Pro excited at 200-240 nm are different from those of Pro--Gly, and assumed that the imide II band (N-H in-plane bending) appears at 1485 and 1515 cm-1 in the trans and cis forms, respectively. Caswell and Spiro (1987), on the other hand, obtained the UV RR spectra of polyproline I with cis imide and polyproline II with trans imide excited around 200-218 nm, and found the imide II band at 1465 cm-1 for the trans form and at 1435 cm-1 for the cis form. With the RR spectra excited in the visible region, it is impossible to discuss the imide mode, since the CH 3 and CH 2 deformation modes are heavily overlapping in this frequency region. In order to examine the two proposals for the imide mode, Takeuchi and Harada (1990b) measured the UV RR spectra of cyclo (Gly-Pro) and linear (Gly-Pro) with the 213 and 240 nm excitation lines, and concluded that both cis and trans imides have the same intrinsic imide II frequency at ~ 1445 cm -1 but, when they are hydrogen bonded to C----O, it shifts up to ~ 1485 cm - 1. They pointed out that the imide II band of X-Pro can serve as a conformation marker for bradykinin and gramicidine S (Takeuchi and Harada, 1990). 5. Markers o f Local Environments

The Raman band of Trp around 1550 cm- 1 is assigned to the W 3 mode (Takeuchi and Harada, 1986). This frequency is considered to be insensitive to hydrogen bonding of the indole ring (Miura et al., 1989). Recently a correlation was found between the W 3 frequencies and the dihedral angle, X2'1, about the bond connecting the indole ring with the Cp atom of the Trp side-chain; a monotonic increase of the W 3 frequency with X2' 1 for 60 ° < Z2' 1 < 120o. Rodgers et al. (1992) pointed out that g 2'1 of f137-Trp of Hb is different from other Trp residues based on the observation of a frequency shift of this band which was assigned with the f137-Trp~Arg mutant Hb. The Raman band of Trp around 1350 cm - 1 is derived from a Fermi resonance between the fundamental of the indole NI--C 8 stretching (WT) and combinations of out-of-plane indole ring bending modes (W25--I-W33 and/or Wza+W29 ) (Miura et al., 1989). The relative intensity of this doublet depends upon the Trp environments. Under very hydrophobic environments a narrow band with a maximum at 1350 cm-1 appears, whereas in the hydrophilic environments the main band shifts to lower frequencies (to ~ 1335 cm- 1) with a shoulder at 1350 cm- 1. The split ring-stretching modes of Tyr (vaa= ,-~ 1615 cm-1, vsb= ,,~ 1600 cm-1) show small frequency shifts in D20, indicating appreciable vibrational coupling with the phenolic OH bending mode. The deuteration shift is larger for Yahthan for Vsa. Their frequencies were correlated with the hydrogen-bond strength for p-cresol as a model compound of Tyr (Hildebrandt et al., 1988), and recently the data were refined to establish the linear relations

Higher order structures of proteins

7

of the vs, and yah frequencies (cm -~) with the association enthalpy of cresol ( - A H in kcal/mol) (Rodgers et al., 1992); vsa= 1617.4-0.31(-AH) and Yah= 1598.5--0.94(--AH). 6. Discernment of Higher Order Structures There is no general marker band which indicates a particular tertiary or quaternary structure, and therefore such bands should be searched in individual proteins. As an example the 218 nm excited RR spectra (Kaminaka et al., 1990) of the fluoride adduct of metHb (HbF) are shown in Fig. 4. Human Hb, an oxygen carrier protein, consists of four subunits (~2fl2), and exhibits cooperativity for oxygen binding. The cooperativity has been interpreted in terms of the chemical equilibrium of two quaternary structures designated as T (low affinity) and R (high affinity) (Perutz, 1979). While the typical molecular structures ofT and R are seen for deoxyHb and oxyHb, respectively, X-ray crystallographic analysis of HbF (Fermi and Perutz, 1977) pointed out that HbF at pH 7.4 and HbF at pH 6.8 in the presence of inositol-hexaphosphate (IHP) serve as a model of R and T, respectively. The UV RR spectra corresponding to the two model molecules are shown by the upper (R) and lower traces (T) in Fig. 4.

MIIHbAF (pHT.4)

l

~-

t

:

°i i!

MelHbAFI-IHPtpH6B) I[ i ;

inl t

Roman Shift

(cm"l)

F[c. 4. The 218 nm excited RR spectra of the fluoride adduct ofmetHb with R structure (upper) and T

structure (lower)(fromKaminakaet al., 1990). The bands of HbF at 1557, 1011, 878 and 756 cm -1 are assigned to W 3, Wt6, W17 and W~a modes (Miura et al., 1989) of Trp, respectively, and the bands at 1585 and 1003 c m - t are assigned to Vaband vx2 of Phe. The bands at 1604 and 1613 cart- t are considered to be an overlapped band of Yahof Tyr with vs, of Phe and of vs, of Tyr with W t of Trp, respectively. In the spectrum ofT (lower), the intensity of the 1011 cm- t band of Trp relative to that of the 1003 cm- ~band of Phe is reduced and the band shape of Wt 7 is symmetric as if a shoulder is present at ~ 883 cm- ~. The W I , frequency is known to be sensitive to hydrogen bonding of Trp (Miura et al., 1988). IV. QUATERNARY STRUCTURES AND ITS DYNAMICS IN H A E M O G L O B I N 1. Quaternary Structure Marker Cooperative oxygen binding of Hb is attributed to the presence of a path through which the binding of 0 2 to an arbitrary haem is communicated to another haem and its oxygen affinity is raised. Questions to be answered are how it is communicated and how 0 2 affinity is varied. There are many hydrogen bonds and salt bridges between subunits. Among them the hydrogen bond between ~42-Tyr and f199-Asp in the deoxy state (see the inset of Fig. 5) was

8

T. KITAGAWA

monitored by NMR spectroscopy (Fung and Ho, 1975); phenolic hydroxy proton gives a resonance at - 9.4 ppm in the deoxy state but it apparently disappears in the oxy state since the hydrogen bond is cleaved and the 1H is exchanged with bulk water. Accordingly, the 1H NMR signal has been used as a T marker (Fung and Ho, 1975).

deoxy Hb(T) ~"Fle'~ "Fie"'

'

His.

I [

r

...His

I

_~,1

I

H,i's" >Fe~, i

I J

"'His

I

J

I

I

,,Fe~

a-subunit

I i

I

I

T.vr .

W

,

1

1

I I

~

I

~/.

_~~

~_ I

~'subunlt

!

I I I

I I

oxy H b (R) I

I I

ct.subunit

~,,fC

I

1

FIG. 5. A schematic model of Hb quaternary structure. T-structure deoxyHb (upper) and R-structure oxyHb (lower). The inset illustrates the typical hydrogen bonds at the ~tl-/~2 interface. The phenolic hydroxy 1H NMR signal of ~t42-Tyr serves as a T marker. (From Fung and Ho, 1975.)

Perutz (1982) proposed, on the basis of the X-ray crystallographic analysis, that stronger interactions occurring in the deoxy (T) state between subunits as well as within a subunit might pull the proximal histidine (F8-His), which is the sole residue bound covalently to the haem group, away from the porphyrin plane because of a "tensed" form of the polypeptide chain. The inter- and intra-subunit interactions are weakened in the oxy (R) state because of a "relaxed" form of the polypeptide chain and F8-His is not pulled away from the haem any more. If this model is correct, the Fe-His(F8) bond might stretch and its bond strength would be weaker in the T than in the R state. Thus, its stretching frequency would be lowered in the T state. In order to examine this idea, Kitagawa et al. (1979) assigned the Fe-His(F8) stretching (v~_m~) RR band of deoxyHb on the basis of the 54Fe isotopic frequency shift, and Nagai et al. (1980) measured the RR spectra of various chemically modified deoxyHbs with varied affinities. The high-affinity and low-affinity deoxyHbs gave the v~_ms band at 220-222 and 214-216 cm-1, respectively, while all other RR bands exhibited negligible differences. This observation is quite consistent with Perutz's assumption (Perutz, 1982) that the hydrogen

Higher order structures of proteins

9

b o n d i n g at the s u b u n i t interface results in g e n e r a t i o n of s o m e strain in the F e - H i s ( F 8 ) b o n d a n d lowers the o x y g e n affinity. 2. Continuous Strain M o d e l T h e m a g n i t u d e of the strain m i g h t be a l t e r e d by a m i n o acid r e p l a c e m e n t o f the g l o b i n a n d it s h o u l d have s o m e c o r r e l a t i o n with o x y g e n affinity. A c c o r d i n g l y , M a t s u k a w a et al. (1981) purified v a r i o u s h u m a n n a t u r a l m u t a n t H b s a n d d e t e r m i n e d the e q u i l i b r i u m c o n s t a n t (K 1) for the first o x y g e n to b i n d the d e o x y H b , a n d also the VFc_nis frequency of the d e o x y state for identical p r e p a r a t i o n s ( M a t s u k a w a et al., 1985). T h e results are s u m m a r i z e d in T a b l e 1

TABLE I. OBSERVED VALUES OF ~/Fe-His AND K 1 FOR SEVERAL HAEMOGLOBINS

NO.* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Haemoglobin (name/conditions) Hb A/pH 7.0, + IHPf Hb A/pH 7.0, +Cl§ Hb A/pH 8.5, + CI Hb A/pH 7.0, -Clll Hb A/pH 8.5, - C l Hb J Capetown/pH 7.0, + IHP Hb J Capetown/pH 7.0, +C1 Hb J Capetown/pH 8.5, +Cl Hb J Capetown/pH 8.5, - C l Hb Chesapeake/pH 7.0, +IHP Hb Chesapeake/pH 7.0, +Cl Hb Chesapeake/pH 8.5, +Cl Hb Chesapeake/pH 8.5, - C I Hb Kansas/pH 7.0, +Cl Hb Kansas/pH 8.5, - C l Hb Yakima/pH 7.0, +IHP Hb Yakima/pH 7.0, +C1 Hb Kempsey/pH 7.0, +IHP Hb Kempsey/pH 7.0, +Cl Hb Hirose/pH 7.0, +IHP Hb Hirose/pH 7.0, +Cl Hb Hirose/pH 8.5, +Cl ct chain fl chain

VFe_His K1 (cm -l) (mmng) 214.5 215 216 216 218 214.5 215 217.5 220 215 216 220 222.5 214 214 216.5 220 217.5 220 218 219 219 223 224

138 70 12.5 6.06 4.9 58 16.7 5.0 1.0 37.1 3.52 1.05 0.60 50 19 26.9 1.02 14.65 1.06 11.5 2.0 1.3 0.6 0.34

L (L=[T]/[R])

c (c=K~JKr)

4.5 x E8~/ 4.35 x E7 5.0 x E3 9.0x E3 9.5 x E2 9.5 x E6 2.0x E5 5.5 x E3

0.0029 0.002 0.008 0.033 0.050 0.0096 0.012 0.04 n.d. 0.0166 0.11 0.33 n.d. n.d. n.d. 0.026 0.36 0.070 0.30 n.d. n.d. n.d.

2.5 x E5 2.5 x E2 1.2 x E1

5.0 x E3 2.0x E1 9.0 x El 2.0 x E1

Kr

KR

138 70 12.5 6.06 5.0 58 16.7 5.0

0.40 0.14 0.10 0.20 0.25 0.40 0.20 0.20

37.1 0.60 3.64 0.40 1.21 0.40

26.9 1.11 17.1 1.33

0.70 0.40 1.20 0.40

* Sample number. f In the presence of 1 mM IHP. :~ En means 10n. § In the presence ofO.1 M NaCI. q[ In the absence of chloride ion.

( M a t s u k a w a et al., 1985), where K 1 is r e p r e s e n t e d in t e r m s of the d i s s o c i a t i o n c o n s t a n t , a n d L, c, K T, a n d Ka d e n o t e M W C p a r a m e t e r s ( M o n o d et al., 1965). It is a p p a r e n t t h a t the VFc_ms frequency b e c o m e s l o w e r as the affinity b e c o m e s lower. This t r e n d is m o r e d e a f l y seen in Fig. 6, where the VFc_nis frequencies are p l o t t e d a g a i n s t K1. T h e s o l i d line represents a theoretical curve ( K i t a g a w a , 1987) which was d e r i v e d o n the a s s u m p t i o n t h a t the s t r o n g e r i n t e r s u b u n i t i n t e r a c t i o n s stabilize further the T-like structure a n d reduce the free energy c h a n g e u p o n o x y g e n b i n d i n g o n the one h a n d , b u t yield the larger strain in the F e - H i s ( F 8 ) b o n d o n the other. Since the t h e o r e t i c a l curve r e p r o d u c e s well the o b s e r v e d results, the strain on the F e - H i s ( F S ) b o n d i m p o s e d b y t b e g l o b i n is e v i d e n t l y o n e o f the i m p o r t a n t factors t h a t c o n t r o l the o x y g e n affinity, a n d this is a c o n t i n u o u s variable. O n e m a y w o n d e r w h e t h e r a small c h a n g e at the p r o t e i n surface is really c o m m u n i c a t e d to the h a e m g r o u p b u r i e d in the g l o b i n a n d alters the o x y g e n affinity. I n o r d e r to a n s w e r this q u e s t i o n , I m a i et al. (1991) c a r r i e d o u t site-directed m u t a g e n e s i s o n ~t42-Tyr o f h u m a n H b , which was n o t e d to form a n i n t e r s u b u n i t h y d r o g e n b o n d o n l y in the d e o x y state (see Fig. 5). T h e i r R R s p e c t r a in the VF~_m~region are s h o w n in Fig. 7. W h e n ~t42-Tyr was r e p l a c e d b y P h e (~t42-Phe), the o x y g e n affinity was raised g r e a t l y from Pso = 45 to 0.6 m m H g in terms o f the

10

T. KITAGAWA

V / cr,n't

,4

i

215

220

225

0.1

I

I

I

I

lllll

I

1.0

I

I

l

....

I

10.0

i

,

,

l

....

I

I00.0

K I / (mmHg)

FIG. 6. The vFc_ms frequencies vs K, plots of various mutant Hbs (from Matsukawa et al., 1985). Closed circles stand for human natural mutant Hbs and individual points are specified by numbers which correspond to the sample numbers in Table 1. Two open circles designated as Phe and His represent 0¢42-Phe and ~42-His mutant Hbs, respectively, obtained through site-directed mutagenesis (see Fig. 7). The solid line indicates the theoretical curve derived under assumption that the energy change of the Fe-His(F8) bond is proportional to the free energy change due to quaternary structure change (Kitagawa, 1987).

half-saturating oxygen pressure, and the VFe_His frequency is shifted up to 220 cm -1 in comparison with 214cm -1 of native deoxyHb A. When ~42-Tyr was replaced by His (~42-His), the hydrogen bonding at the subunit interface becomes medium and in accord, the oxygen affinity is moderate (Pso = 1.4 mmHg) and the vre_ms band is observed between the two typical ones. It is emphasized that, when the V~e_Hi~frequencies and K 1 determined for these synthetic mutant Hbs (Imai et al., 1991) are plotted in Fig. 6, they fall on the curve obtained previously for the natural mutant Hbs, as designated by open circles in Fig. 6. Thus, we can have more confidence that a small change at the subunit interface is conveyed to the haem group through the Fe-His(F8) bond. 3. Time-Resolved U V R R Spectra It is known that the carbon monoxide adduct ofdeoxyHb (COHb) adopts the high affinity quaternary structure. CO can be photodissociated in 80 fsec (Martin et al., 1983), but relaxation of the protein structure from R to T is much delayed. The protein structural changes following the photodissociation of COHb were pursued with time-resolved UV RR spectroscopy (Kaminaka et al., 1990). The layout of the observation system is illustrated in Fig. 8(A). The excitation light for Raman scattering was generated by Raman shifting the fourth harmonic of a Nd:YAG laser. The fundamental, second and fourth harmonics of the Nd:YAG laser were focused into a lm H2 cell (7.5 atm.) and the second anti-Stokes line (218 nm) was selected by a beam separator constructed with rectangular and Pellin-Broca prisms. The laser power for the 218 nm line was 100-120/~J/pulse at the sample, but its flux photon density was lowered to 3 mJ/(cm2pulse) by the system illustrated in Fig. 2(A). The pump beam for photodissociating COHb was obtained from an N 2 laser-pumped dye laser, which was operated at 419 nm with a dye of bis-MSB, and its power was 180/~J/pulse at the sample. The delay time (Atd) of the Raman probe pulse (8 nsec width) from the pump pulse (8 nsec width) was controlled by a pulse generator (PG in Fig. 8A) system. BPG in Fig. 8A represents a 10 Hz PG consisting of a 1 MHz quartz oscillator which generates two pulses; one triggers PG1 and the other triggers PG2 to fire each laser at the desired time. The actual delay time was determined by putting the partially reflected light of the pump and probe pulses into the photodiodes (PD1 and PD2) and by monitoring their output with an oscilloscope. The two beams were introduced to the sample illuminating chamber, which is illustrated in Fig. 8(B). The sample is circulated by a peristaltic pump and illuminated at the wire-guided flow device (Fig. 2(B)) placed in the chamber. The back scattered light at 120° was dispersed with a 1.26m single-monochromator equipped with a 2400 grooves/mm holographic grating (used in the second order) and detected with a UV sensitive photomultiplier (Hamamatsu

Higher order structures of proteins

11

o('4 ~,

/ " (A)

~

,¢

(A')

,3 1'9o.1 A

~

/

V PSO= 0.6

42(x-Phe + I HP P.50" 1.0

(B')

42or-His

P~o" 1.4

c)

42o~-HIs + IHP PSO" 15

C')

Hb A PSO = 4.7

HbA+IHP P50" 45 i

I

400

i

I

3OO

I

I

i

200

RAMAN SHIFT/cm -I FIG. 7. The RR spectra in the Fe-His stretching region of mutant deoxyHbs obtained through site-directed mutagenesis. (A) a42-Phe, (B) =42-His, (C) native HbA. (A'), (B'), and (C') were observed in the presence of 2 mM IHP for (A), (B) and (C), respectively. In all cases, Hb concentration is 100/z~l on haem bases in 0.05 M bis-Tris (pH 7.4) containing 0.1 M CI-. Pso denotes the half-saturating oxygen pressure in terms of mmHg. Excitation: 441.6 nm (from Imai et al., 1991).

RI66UH). For shorter delay time (Ata<20 nsec), two pulses with different wavelengths obtained from the output of the H2 Raman shifter were separated by a device shown in Fig. 8(C), and the probe pulse was delayed by making its path length longer (0.033 nsec/cm). Time-resolved UV RR spectra (Kaminaka et al., 1990) for the delay time between A t a = - 1 0 0 and +500/zsec are displayed in Fig. 9 (1750-1350cm -~) and in Fig. 10 (1050--650 cm- 1). The band at 1003 cm- ~is assigned to Phe residues but all other bands are assignable to Trp residues. The spectrum for At a = - 100/asec was the same as that observed without the pump beam. While, for A t a = 10 nsec, the 436 nm pulse obtained from the same Nd: YAG laser was used as the pump beam and A t a was generated by the optical delay, its spectrum is in agreement with that for A t a = - 100 #see. Although, in the initial report of time-resolved UV RR spectra of COHb, Dasgupta et al. (1986) reported a change of protein structure within 7 nsec of photodissociation, which contradicted the results shown in Figs 9 and 10, and the results were withdrawn afterwards by successors in the same lab (Suet al., 1989).

12

(A)

T. KITAGAWA

IOHzOIc.

I

DeloyPuI.Gene. r'="""~,

Dye L o | e r r~L. u o B , t I'~lJ" m~DI

=,.~_ . . l'l'K ~ I I

419nm

O|¢lllo|cope

(B)

~

tOgas~ 7

fromperistalticpump (a)

1

Nd-YAG Loser I

~. ~

R a m ~ $hlt~r

gll'7~Snm

Delay Pul.Glme. (C)

~ ~ I. .... (d) 419n~(h)

( N=-Dye)

218nm probe

~. _J

/~

(e)

u...

T

....

cogos,J ;

to peristaltic pump

FIG. 8. Schematic diagram of the time-resolved UV RR measurement system used by the author's group. (A) Layout of the system; PG: pulse generator; PD: photodiode. (B) The sample illuminating chamber used for Hb experiments; (a) upper lid, (b) wire-guided sample-flowing device, (c) a box made of quartz, (d) an adapter for the bottom lid which serves as a small reservoir of the sample solution, (e) bottom lid, (f) window for watching, (g) probe light, (h) pump light. (C) A device for separation of the output light of the H 2 Raman shifter (from Kaminaka et al., 1990).

extn.217.enm

:~, ,¢

AfterCOphotodissoc.~1

~

50ps

~

lOOp.,

*.IOns i

'

lOps

~

~

200p o

20ps

5OOp RamanShift(cm")

RamanShift(cm"u)

FIG. 9. Time-resolved UV RR spectra of photodissociated COHb A in the 1750--1350 cm - 1 region. The delay time of the probe light (217.8 nm) from, the pump light (419 nm except for Atd = 10 nsec for which 436 nm was used) is specified at the left side of each spectrum. The spectrum for A t d = - 100/~sec is confirmed to be the same as that obtained without the pump beam (from Kaminaka et al., 1990).

Higher order structures of proteins

13

The spectrum for At a = 5 #sec (not shown) was close to that for At d = 10 psec. The intensity of the bands at 1613, 1011 and 878cm -1 relative to nearby bands are varied for At d = 10-20/asec but are restored for longer Atd. Since the S I N ratios of the spectra are not sufficiently high and the changes are small, the results may not justify a discussion of the Atd-dependent spectral changes. Therefore, completely independent experiments with different preparations of the protein were carried out for only two typical values of At d, for which the spectral accumulation time was made twice as long as the previous one. The results are shown in Fig. 11, where the spectra for At d = - 100 #sec (A) and At d = 20/~sec (B) are displayed. In these spectra with improved S I N ratios, the intensity reduction of the 101 ! cm - x band and the frequency shift of the 878 c m - 1 band noticed for At d = 10-20 #sec in Fig. 10 are reproduced well. Accordingly, the RR spectra for other At a psec shown in Fig. 10 are also considered to be reliable and used for further analysis. In order to see the time dependence of spectral change more clearly, the relative intensity of the two bands at 878 and 883 c m - *, Ias3/Ia7 s, are plotted against the delay time in the inset of Fig. 11. The horizontal line indicates the value at At d = l0 nsec. Error bars were estimated from the highest and lowest values in eight measurements. The relative intensity reached the maximum at At d = 20 #sec and decreased to its original value. This restoration is due to regeneration of the CO-bound form in the sample illuminating chamber filled with CO. It can be concluded from this experiment that the protein structural change starts at - 5 psec after photolysis, but the end time of this change could not be determined.

ectn.217.8nm

~_/

After COphotodlsscx

-lOOps

~lOre

lOps

20., Romon Shift (cm "t )

Roman Shift (cm-')

FIG. 10. Time-resolved UV RR spectra of photodissociated COHb A in the 1050-650 cm-1 region. Experimental conditions are the same as those for Fig. 9 (from Kaminaka et al., 1990).

There are three Trp residues per ctfl dimer of Hb A and one of them (fl37) undergoes a change of status upon change of the quaternary structure, while two other Trp residues (ct14 and fll 5) are contained in the 0t-helix and remain unchanged upon the quaternary structure change (Baldwin and Clothia, 1979). The f137-Trp is located at the ~t1-f12 subunit interface, as will be explained in more detail later, where this residue is in contact with 0tl40-Tyr in T but is freed from it in R (Baldwin and Clothia, 1979). The UV absorption differences between the Tand R-structure Hbs have been attributed to environmental changes of f137-Trp and ~t42-Tyr (Perutz et al., 1974). Furthermore, recently Spiro et al. (1991) observed a change in the W 3 band of Trp for the spectrum of At a = 10 ltsec of photodissociated C O H b , and ascribed it to

14

T. KITAGAWA

After CO photodissociation

extn. 21Z8nm

(A)-IOOps

I

Ice'a/Ie~'e

1.5

1.0

0.5 lose (O-IOns) ~I'-.--~

I

i0"z

lO.4 Time/sac

O9

(B) 20ps

Raman Shift (cm") FIG. 11. Time-resolved UV RR spectra of photodissociated COHb A obtained in separate experiments with longer accumulation time. (A) Atd= - 100 #sec, (B) Atd= 20 #sec. The inset figure plots the intensity at 883 cm- 1relativeto that at 878 cm- ' against the delay time. The horizontal line indicates the value for Atd= 10 nsec. The error bars wereestimated from the maximumand minimum values in the eight independent measurements (from Kaminaka et al., 1990).

f137-Trp on the basis of the measurements on f137-Trp~Arg mutant (Hb Rothschild). Accordingly, the UV RR spectral change observed in Fig. 11 is presumed to arise from f137Trp.

4. Bohr Effect and a Mechanism of Quaternary Structure Change To circumvent the rebinding problem, a new sample flowing system which allows the use of N2-filled sample-illuminating chamber was constructed (Kaminaka and Kitagawa, 1992). The spectra obtained with it for At o = + 200/~sec and - 100/~sec are displayed in Fig. 12. The spectrum for At o = - 100 #sec (B) is in agreement with the spectrum of C O H b shown in Fig. l l(A), indicating that C O is not chemically dissociated in the N2-filled chamber before illumination of the p u m p beam. The spectrum for Atd=200/tsec (A) differs from the spectrum for Ata=200/~sec observed under C O atmosphere (Fig. 10), but is close to the spectrum for At a = 20 #sec in Fig. 1 I(B). Therefore, it is clear that recombination of C O does not take place in the sample chamber. The intensity of the T r p band at 1011 c m - 1 relative to the Phe band at 1003 c m - 1 is different between At o = - 100 #sec and + 200/~sec. The relative intensity of these two bands was scarcely varied by the increase of the laser power and also by its decrease to half and, therefore, it would not be caused by depletion of the ground state molecules (Teraoka et al., 1990). In haem proteins, the energy transfer from aromatic residues to haem is efficient, and accordingly the de-excitation of aromatic residues is generally fast (Harmon et al., 1990). It is unexpected that the W 17 frequency of T r p is unaltered between the two spectra in Fig. 12, but no frequency shift of W17 between d e o x y H b and C O H b (Spiro et al., 1991 ) was confirmed by repeated measurements (by 20 scans) ( K a m i n a k a and Kitagawa, 1992). The intensities ofthe Trp band at 1011 c m - ~ relative to that of the Phe band at 1003 cm - 1 (11o11/11oo3) obtained with the N2-filled chamber are plotted against At d in Fig. 13 ( K a m i n a k a and Kitagawa, 1992), where panels A and B were obtained for the C O H b solutions at p H 7.4 and p H 5.8, respectively. The broken lines indicate the value for

Higher order structures of proteins

15

CO photodlssoclaflon

(N= =rnosp.er,

(A] 200ps After ~tl

(B) l O O p s Before

o o h.

I

Roman Shift/cm" FIG, i 2. Time-resolved UV RR spectra of photodissociated COHb A in the 1050-800 cm- 1 region observed with the N2-filled sample chamber. (A) Atd----200~sec, (B) A t a = - 1 0 0 ~ s e c (from Kaminaka and Kitagawa, 1992).

(A)

1.5 ~

pH 7.4

13 ' - - t

0.7~

(B)

.......................

i 5

~ I0

e 20

i 50

lOOps

I 500 (ps)

i e I00 200

1.5

pH 5.8 -lOOps

1.3

t = 0.9

tt

0.7 t

l

I

I

I

I0 20 50 I00 200 500 (ps) FIG. 13. The intensity of the Trp band at 1011 cm-s relative to that of the Phe band at 1003 c m - t plotted against the delay time. The time-resolved UV RR spectra were observed with the N2-filled sample chamber and with the 218 nm line as a probe light. The broken line indicates the value for Aid=- 100/~sec. (A) The solution at pH 7.4, (B) The solution at pH 5.8 (from Kaminaka and Kitagawa, 1992).

16

T. KITAGAWA

N,-'~ ~-~ N ~

C~(~139Lys) I~

,,C~ (~89 His)

,~-helix(C-term) Ca (a 14oTyr)

FG-eorner iv" ~

\ / , f Ca( ~ A l a ) ;

F-helix

\ /

/ I

(

/

/

Ca(a87His)

/ \ \ // )---/

, " r .... ". C~(~37Trp) /

'~

"~

:

~"

C-he,lx

~-Heme

FtG. 14. The structure of deoxyHbnear ct87-ct89,~139--ct140,and fl37residues taken fromcomputer graphics (original, Fermi et al., 1984). A t d = - 100/~sec, while the solid lines were drawn to correlate the observed points smoothly.

At pH 7.4, the spectral change starts at 5/~sec in agreement with the results shown in the inset of Fig. 11. The occurrence of the R ~ T transition in this time region is also consistent with the results from the transient absorption experiments (Hofrichter et al., 1983). The 11o 11/11oo3 value changes smoothly until A t d = 20 #sec and afterwards remains constant. Although Suet al. (1989) reported the presence of an intermediate state at At d = 5 btsec from the UV RR measurements of Trp and Tyr bands excited at 228.7 nm, the plots shown in Fig. 13(A) suggest a simple two state change, so far ~ls this Trp band is concerned. When 0 2 is bound to deoxyHb, protons are dissociated from the globin. This is called the alkaline Bohr effect (Bohr et al., 1904), which plays a substantial role in the O2/CO 2 exchange in tissues and has been known as "lowering of 0 2 affinity upon acidification" (Wyman, 1964); deoxyHb + 402,---,Hb(402) + 2.4H ÷ Protonation of a few amino acid residues (Bohr residues) at acidic conditions causes lowering of the 0 2 affinity. To understand this hetrotropic allosteric effect, UV RR spectra were measured at pH 5.8 (Kaminaka and Kitagawa, 1992) where Bohr residues are partially protonated. The 218 nm excited RR spectra for At d = - 100 and 500/~sec at pH 5.8 were the same as the corresponding spectra at pH 7.4, indicating that the structural change is not greatly altered by the protonation of Bohr residues. However, when 11o 11/11oo3 w e r e plotted against Atd, it was different from that at pH 7.4 as illustrated by Fig. 13(B). The relative intensity had already changed at Ata = 10 #sec and gave the same value until Atd = 500 #sec. The 11011/11003 values at A t d = - 100/Lsec and at Atd=500/~sec are scarcely altered by protonation of Bohr residues, but the dynamics were altered. The acceleration of the R to T transition at acidic pH is consistent with the fact that the R-T equilibrium is biased to T in acidic conditions. Among several kinds of Bohr residues, this dynamic difference was deduced to arise from protonation of ct89-His (Kaminaka and Kitagawa, 1992), which has the p K a values of 7.2 for deoxyHb and 5.6 for C O H b (Ohe and Kajita, 1980). As illustrated in Fig. 14 (Fermi et al., 1984), neutral ct89-His can be hydrogen bonded with e-NH~ of ~139-Lys, but they would

Higher order structures of proteins

17

repel each other u p o n p r o t o n a t i o n of ~89-His. The possible m o v e m e n t of ~139-Lys u p o n p r o t o n a t i o n to ~89-His would induce some m o v e m e n t of the next residue, ~ 140-Tyr, which is k n o w n to interact with f137-Trp (Baldwin and Clothia, 1979). The ~89-His is located at the end of the F helix near the proximal His (F8, ~87) (see Fig. 14). Time-resolved visible R R spectra covering the •Fe---His b a n d (Scott and Friedman, 1984) d e m o n s t r a t e d that the F e - H i s ( F 8 ) b o n d changes continuously in the time range from 10 nsec to 10/~sec after photolysis, a l t h o u g h the protein moiety exhibited little change during that time range. W h e n the m o v e m e n t of ~87-His is completed, ~89-His, ~139-Lys and ~140-Tyr are affected in turn, and finally f137-Trp is altered. In this way a change in the haem ligation in the ~ subunit can be c o m m u n i c a t e d to the fl subunit t h r o u g h c o n f o r m a t i o n changes, and this was elucidated by time-resolved U V R R spectroscopy. This view of the cooperativity mechanism is quite consistent with the recent observation of non-cooperative oxygen binding and no Bohr effect for single crystal H b (Mazzarelli e t al., 1991) in which the intersubunit salt-bridges as well as intrasubunit tertiary structure are retained in the T structure by the lattice forces of crystal even after oxygen binding. ACKNOWLEDGEMENTS The a u t h o r is indebted to Drs Shoji K a m i n a k a and Takashi O g u r a of the Institute for Molecular Science, for their c o o p e r a t i o n on U V R R studies. This study was supported by Grant-in-Aids for Scientific Research, of the Ministry of Education, Science and Culture (02454157). REFERENCES AMES,J. B., BOLTON,S. R., NETTO,M. M, and MATHIES,R. A. (1990) J. Am. Chem. Soc. 112, 9007. ASHER,S. A., JOHNSON,C. R. and MURTAUGH,J. (1983) Rev. Sci. lnstrum. 54, 1657. ASHER,S. A., LUDWIG,M. and JOHNSON,C. R. (1986) J. Am. Chem. Soc. 108, 3186. BALDWIN,J. and CHOTHIA,C. (1979) J. molec. Biol. 129, 175. BOHR,C., HASSELBACH,K. and KROGH.A. (1904) Scand. arch. Physiol. 16, 402. BROWN,K. G., BROWN,E. B. and PERSON,W. B. (1977) J. Am. Chem. Soc. 99, 3128. CAGEY,P. R. (1982) Biochemical Applications of Raman and Resonance Raman Spectroscopies, Academic Press. CASWELL,D. S. and SPmo, T. G. (1986) J. Am. Chem. Soc. 108, 6470. CASWELL,D. S. and Spmo, T. G. (1987) J. Am. Chem. Soc. 109, 2796. CLARK,R. J. H. and HESTER,R. E. (eds.) (1986) Raman Spectroscopy of Biological Molecules, Wiley and Sons. COPELAND,R. A. and SPmo, T. G. (1985b) Biochemistry 24, 4960. COPELAND,R. A., DASGUPTA,S. and SP1RO,T. G. (1985) J. Am. Chem. Soc. 107, 3370. COPELAND,R. A. and SPIRO,T. G. (1986a) J. phys. Chem. 90, 6648 (1986). COPELAND,R. A. and SPIRO,T. G. (1986b) J. Am, Chem. Soc. 108, 1281. COPELAND,R. A. and SPIRO,T. G. (1987) Biochemistry 26, 2134. DASGUPTA,S., COPELAND,R. A. and SPIRO,T. G. (1986) J. biol. Chem. 261, 10960. DUDIK,J. M., JOHNSON,C. R. and ASHER,S. A. (1985a) J. phys. Chem. 89, 3805. DUDIK,J. M., JOHNSON,C. R. and ASHER,S. A. (1985b) J. chem. Phys. 82, 1732. FERMI,G. and PERUTZ,M. F. (1977) J. molec. Biol. 114, 421. FERMI,G., PERUTZ,M. F., SHAANAN,B. and FOURME,R. (1984) J. molec. Biol. 175, 159. FODOR,S. P. A., RAVA,R. P., COPELAND,R. A. and Spmo, T. G. (1986) J. Raman Spectrosc. 17, 471. FUNG, L. W.-M. and Ho, C. (1975) Biochemistry 14, 2526. GERRITY,O. P., ZIEGLER,L. D., KELLY,P. B., DESIDERIO,R. A. and HUDSON,B. (1986) J. chem. Phys. 83, 3209. HARMON,P. A., TERAOKA,J. and ASI-mR,S. A. (1990) J. Am. Chem. Soc. 112, 8789. HILDEBRANDT,P. G., COPELAND,R. A., SPIRO,T. G., OTLEWSKI,J., LASKOWSKI,M. Jr and PRENDERGAST,F. G. (1988) Biochemistry 27, 5426. HOFRICHTER,J., SOMMER,J. H., HENRY,E. R. and EATON,W. A. (1983) Proc. natn. Acad. Sci. U.S.A. 80, 2235. IMAI,K., FUSHITANI,K., MIYAZAKI,G., ISHIMORI,K., KITAGAWA,T., WADA,Y., MORIMOTO,H., MORISHIMA,I., SHIn, D. T. and TAME,J. (1991)J. molec. Biol. 218, 769. JOHNSON,C. R., LUDWIG,M., O'DONNELL,S. and ASHER,S. A. (1984) J. Am. Chem. Soc. 106, 5008. JOHNSON,C. R., LUDW1G,M. and ASHER,S. A. (1986) J. Am. Chem. Soc. 108, 905. JOHNSON,C. R. and ASHER,S. A. (1987) J. Raman Spectrosc. 18, 345. JONES,C. M., DEVITO,V. L., HARMON,P. A. and ASHER,S. A. (1987) Appl. Spectrosc. 41, 1268. KAMINAKA,S., OGURA,T. and KITAGAWA,T. (1990) J. Am. Chem. Soc. 112, 23. KAMI~qAKA,S. and KXTAGAWA,T. (1992) J. Am. Chem. Soc., in press. KITAGAWA,T., NAGAI,K. and TSUBArd,M. (1979) FEBS Lett. 104, 376. KITAGAWA,T. (1987) Pure appl. Chem. 59, 1285. KRIMM,S. and BANDEKAR,J. (1986) Adv. Protein Chem. 38, 181. LIPPERT,J. L., TYMINSKI,D. and DESMEULES,P. J. (1976) J. Am. Chem. Soc. 98, 7075. MARTIN,J. L., MIGUS,A., POYART,C., LECARPENTIER,Y., ASTIER,R. and ANTO~rETTI,A. (1983) Proc. natn. Acad. Sci. U.S.A. 80, 173. MATSUKAWA,S., MAWATARI,K., SmMOKAWA,Y. and YONEYAMA,Y. (1981) J. molec. Biol. 150, 615.

18

T. KITAGAWA

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