Apr 2, 1987 - I. "'F n.m.r. spectra of (a) F-Quene I and (b) 2 HFPD in the anaesthetized ... nuclear Overhauser spectroscopy; NOE. nuclear Overhauser effect.
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BIOCHEMICAL SOCIETY TRANSACTIONS
10pp.m
CF3
OH
CF3 \ / ,
\
C = O + H,O
C
/
CF3
/
CF3
-
CF,
OH \
t
\
I
OH
/
C CF,
\
0
Fig. I . "'F n.m.r. spectra of ( a ) F-Quene I and ( b ) 2 HFPD in the anaesthetized rat liver The equation shows the reversible addition of water to hexafluoroacetone yielding HFPD. The chemical shift of HFPD is given referenced to trifluoroacetate (TFA). For each spectrum lo4 scans were acquired (17min).
We arc grateful to the Staff o f the M.R.C. Biomedical N M R Centre, N.I.M.R., Mill Hill for n.m.r. facilities and help in the construction and design of probes. J.S.B. holds an M.R.C. Research Studentship.
Rogers. J . . Hesketh, R. T.. Smith, G. A. & Metcalfe. J . C . (1983) J . Biol. Chenl. 258. 5994 5997 Tsien. R. Y . (1980) Biochcniisrrv 19, 2396 2404
Iles, R. A., Stevens, A. N., Griffiths. J. R. & Morris, P. G . (1985) Biochmi. J . 229, I 4 I 15 I
Received 2nd April 1987
N.m.r. study of acylphosphatase V. SAUDEK*, M. R. WORMALDT, R. J. P. WILLIAMST and G. RAMPONIS * Institute of' Macromolecular Chemistry, Prague, Cxchoslovakiu, jlnorganic Chemistry Laboratory, Univrrsity of' OxfOrd, U .K . and 1Institute of Biological Chemistry. University of' Florence, Italy Acylphosphatase (EC 3.6.1.7), an enzyme of about 100 residues, catalyses the hydrolysis of organic acylphosphates according to the following equation (Cappugi et al., 1980): RCOOPO:
+H,Od
RCOOH
+ HPO;
I t has been isolated from various tissues (heart, brain, skeletal muscle, liver, erythrocytes) of several vertebrates Abbreviations used: n.m.r.. nuclear magnetic resonance; COSY, two-dimenstional correlated spectroscopy; NOESY, two-dimensional nuclear Overhauser spectroscopy; NOE. nuclear Overhauser effect.
(human, horse, pig, ox, duck, turkey, trout, water-snake). The reaction with metabolically important substrates such as 1,3-diphosphoglycerate or carbamylphosphate is thought to be involved in the metabolic pathways regulation (Cappugi rt al., 1980). Although the amino acid sequence of acylphosphatase from several sources has been determined (summarized in, e.g. Camici et al., 1986), little is known about the overall structure. The enzyme is highly conserved and consists of one chain only. There is one cysteine in the sequence which appears to be cross-linked via an S-S bridge to glutathione during isolation (Camici et al., 1986). Circular-dichroism studies indicate that the enzyme contains some /?-sheet structure and possibly a small amount of a-helix (Camici et al., 1981). Crystallization of the enzyme has not yet produced samples suitable for X-ray diffraction studies. I n this communication we present the first results of a structural study of horse muscle acylphosphatase (sequence and isolation; Cappugi et al., 1980) using two-dimensional I987
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622nd ME E T ING, LEICESTER
Va I
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(b)
~
1
c
-
~ 20
~
~
~ c V V c v V 4K0 N T S K c T V T C Q V Q C P E E K V 60 N S f l K S U L S
K V C S P S S R I D R T N80F S N C K T I S K L E Y S N F S V98 R Y ~ g
Fig. I . ( a ) A schrmatic representution of' the,four-stranded fi-sheet,found in horse muscle acylphosphatasc~.The arrows indicate the strueturall-v important hockhonc NO Es. ( h ) The sequenee of' horse muscle acylphosphatase
Vol. 15
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BlOCH EM ICAL SOCIETY TRANSACTIONS
874 nuclear magnetic resonance (n.m.r.) spectroscopy. COSY, relayed COSY and NOESY experiments (Wuthrich, 1986) were performed at 500 MHz on 4 mM samples of enzyme at pH 5.2 in water or deuterated water. Standard assignment procedures were followed (Wuthrich, 1986). First-stage assignment (assignment of resonances to particular residue types) has been completed for about 80% of the residues; second stage (assignment of resonances to sequence specific amino acids) for about 50%. The NOESY experiments have also provided information on the spatial proximity of sequentially distant residues, thus allowing the determination of regions of regular secondary structure. Four regions of the enzyme (residues 7-14, 36-41, 47-53 and 77-86) show strong sequential CH,-NH NOES (approximate distance 0.2 nm), very small intra-residue CH,-NH NOES (approximate distance > 0.4 nm) and coupling constant of CH,-NH doublet 'J(CH,-NH), of about 8 Hz (torsion angle 4 approx. 8G180deg.) indicating extended conformations of the backbone in these regions. Analysis of the inter-strand CH,-CH,, NH-NH and CHI-NH found between these regions (Fig. 1) reveals the presence of a four-stranded anti-parallel /&sheet, with a /$-bulge at residue 82. A turn between residues 42 and 46 is inferred from the relative positions of the immediately preceding and following sequences (Fig. I ) . The N H / N D exchange rates of the backbone amide protons in the residues assigned to the []-sheet are very slow, showing the presence of strong hydrogen bonding.
A short sequence. displaying some of the characteristic NOEs of a helix, has been found between residues 56 and 59. One side of the /I-sheet consists mainly of hydrophobic residues, the other of hydrophillic ones. A large number of NOEs are found between the hydrophobic residues and hydrophillic residues in other regions of the enzyme. In particular, most of the aromatic side-chains are found to be in close proximity to each other. The variation in intensity of some of the sequential NOES within the /I-sheet indicate that considerable distortions from the planar structure occur. This suggests that the overall conformation may be based on a /I-barrel, the fi-sheet forming part of the surface. The rest of the molecule would then fold to complete the barrel and envelop the hydrophobic core. A more detailed structure is underway. Camici. G.. Manao. G.. Cappugi. G. & Ramponi, G . (19x1) Physiol. Chcm. Pl1y.v. 13. 267 273 Camici, G., Manao. G., Modesti. A,. Stefani. M., Berti, A,, Cappugi. G. & Ramponi. G. (1986) Iiul. J . Biochem. 35. I IS Cappugi. G.. Manao. G.. Camici. G. & Ramponi. G. (1980) J . Bid. Ch1.m. 255. 6868 6874 Wiithrich. K . (1986) N M R of' Protcins cmd Nuc,/cic, Acids. Wiley and Sons
Received 27 March 1987
The interaction of the SH,/SH, region of the myosin heavy chain with actin A. JANE GRIFFITHS, BARRY A. LEVINE* and
IAN P. TRAYER Dqmrtnicnt of Biochemisrr!3. University of Birmingham. PO Box 363, Edghuston. Birmingham BI.5 2TT und *Inorgunic Chcmisrr), Luhoratorjq, Universitjx of O.\-ford, South Purks Road, OxfOrd O X 1 3QR, U . K . The mechanism and regulation of muscle contraction is an excellent example of how proteins respond to external stimuli and transmit information through macromolecular complexes. In muscle, this process involves a dynamic interaction between actin. myosin and nucleotides. A molecular description of this system therefore requires a structural definition of the sites of interaction of these species. To facilitate the investigation of these surfaces of interaction one can exploit the fact that both myosin and actin can be cleaved into smaller function-related segments that can be analysed by n.m.r. for their dynamic and conformational features. I t is clear, for example. that a major site of interaction with actin occurs on the C-terminal 20 kDa domain of the myosin head region (Chaussepied et al., 1986). This fragment. which contains the two fast-reacting SH, and SH2 thiol groups (Cys-705 and Cys-695 respectively), interacts with actin independently of the presence of ATP. Larger fragments, containing at least an additional 60 amino acid residues at the N-terminus, interact with actin in an ATPsensitive manner (Chaussepied e l ul., 1987; Griffiths & Trayer, 1987). Subsequent studies have shown that a nitroxyl spin-label located on the SH, thiol of the myosin head region will perturb resonances in the ' H n.m.r. spectrum of the N-terminal region of actin (Moir ct ul., 1987). Since this region around the SH, 'SH, thiol groups of the myosin heavy chain is strongly conserved in the sequences of seven different myosins, ranging from Dictyostellim discoides to Abbreviations used: n.m.r., nuclear magnetic resonance; CNBr. cyanogen bromide.
rabbit, it suggests it may play an important role in the structureifunction of myosin. and was therefore chosen for further study. A 30-residue peptide, incorporating residues 687--716 of the rabbit skeletal myosin heavy chain. was therefore synthesized: N- EhX7 LV L,,,, H EL RC,,, NGV LE,,,, GI R I T7,,5 RKGFP7I"SRILY7,,A-C. Thr was inserted instead of Cys at position 705 (as occurs naturally in the Dict~~ostellium sequence) to simplify the purification procedure. The synthesis (carried out by the Macromolecular Analysis Service of the University of Birmingham) was by the conventional solid-phase route and the peptide was then purified by gel filtration and ion-exchange chromatography. The binding of this peptide to actin and to the N-terminal CNBr-fragment of actin (containing residues 1-44) could be readily demonstrated by n.m.r. Selective broadening of reyonances arising from specific amino acids occurred. In particular, the aromatic protons of Tyr,,, and Phe,,,,. Arg 6-CH2,and methyl protons of Leu, Ile and/or Val were especially broadened. This suggested that binding may be occurring at the C-terminal end of the peptide. A second peptide was therefore synthesized containing residues 70 1-71 7 (7 I7 = Asp). Studies with this peptide have confirmed the above observations and are helping to identify precisely the residues involved in actin binding to this region of myosin. Care has to be taken when working with these peptides as they appear to be readily oxidized into disulphide dimers. The resulting dimers seem to cross-link actin filaments causing precipitation. This work is supported by grants from the Science and Engineering Research Council and the Medical Research Council. Chausscpied. P., Mornet, D.. Audemard. E.. Kassab, R.. Goodearl. A. J . . Levine. B. A. & Trayer. I . P. (1986) Biochmisrrj. 25. 4540 4547
1987
