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
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
