Download The Number of Protein Subunits Per Helix Turn in Narcissus Mosaic

J. gen. Virol. (1985), 66, 177-179. Printed & Great Britain
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Key words: NMV[ potexvirus/protein subunits
The Number of Protein Subunits Per Helix Turn in Narcissus Mosaic
Virus Particles
By J. N. L O W , P. T O L L I N AND H. R. W I L S O N 1.
Carnegie Laboratory of Physics, University o f Dundee, Dundee D D1 4 H N and 1Physics Department,
University of Stirling, Stirling FK9 4LA, U.K.
(Accepted 15 October 1984)
SUMMARY
By combining the results of Fourier transform calculations from digitized electron
micrographs and molecular volume calculations based on the amino acid composition
and RNA content of narcissus mosaic virus particles, firm evidence that there are 8-8
protein subunits per turn of the helix in the virus particles was obtained.
Narcissus mosaic virus (NMV) is a potexvirus and has elongated flexuous particles, with a
length of about 550 nm and a diameter of about 13 nm (Tollin et al., 1967). X-ray diffraction
studies of orientated virus particles can be interpreted in terms of a helical arrangement of
protein sabunits, with 5q - 1 subunits in five turns of the helix, where q is an integer lying in the
range 7 <~ q <~ 10 (Tollin et al., 1968). The pitch of the helix varies from 3.6 nm when the water
content of the specimen is high to 3.3 nm in the dry specimen.
Optical diffraction patterns from electron micrographs of NMV particles can also be
interpreted on the basis of a helical arrangement of protein subunits. The first studies (ToUin et
al., 1975) suggested that the value of q was 7, but a re-evaluation of the optical diffraction
patterns suggested that q was 9 (Bancroft et al., 1980). Although the X-ray diffraction evidence
(Tollin et al., 1968) also slightly favoured a q value of 9, we have now combined the results of
Fourier transform calculations based on digitization of the electron micrographs (Low, 1982),
and molecular volume calculations based on the amino acid composition of the N M V protein
and the R N A content of the virus which give unambiguous evidence in favour of q = 9.
The optical diffraction patterns only give information about the amplitudes in the Fourier
transform whereas the calculated transform from digitized images gives the phases of the
transform as well. This makes it possible to distinguish between an even and an odd Bessel
function because the phase difference across the meridian (see Fig. 1 of Tollin et al., 1975) for an
even Bessel function is zero, but 180° for an odd Bessel function. I f we consider the possible
numbers of subunits per turn in the range 7 ~< q ~< 10 (Table I), then for 9.8 or 7-8 subunits per
turn, the Bessel function contributing to the first layer-line is even, whilst for 8-8 or 6.8 it is odd.
The calculations (Low, 1982) show that the phase difference across the meridian is close to 180°
and hence favours q --- 7 or q = 9.
Molecular volume calculations based on amino acid volumes and their packing density in
proteins, combined with lattice volume determinations from X-ray diffraction patterns of dry
orientated virus particles can also be used to give an estimate of the number of protein subunits
per turn of the helix (Makowski & Caspar, 1978). The amino acids in the interiors of protein
molecules are closely packed (Richards, 1974) and the volume occupied by a particular amino
acid in the interior of a protein is constant to within a few percent (Chothia, 1975). Furthermore,
the amino acid volume is the same at intersubunit contacts in protein assemblies as in the
protein interior (Chothia & Janin, 1975). In a dry specimen of orientated elongated viruses the
particles interlock and pack close together in a hexagonal arrangement, and in such specimens
the conditions are similar to those in the interior and contact regions of protein molecules.
The amino acid composition of NMV has been determined (Short, 1982) and X-ray
diffraction from dry specimens of NMV shows that the particles pack in a hexagonal
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Table 1. The variation in the order of the Bessel function on the first layer-line of the optical
diffraction pattern, and of the ratio of the effective lattice volume available for the protein and nucleic
acid to the estimated total volume of protein and nucleic acid, with the number of protein subunits per
turn of the helix
Number of protein subunits
per turn of the helix
9-8
8.8
7.8
6.8
Order of Bessel function
on the 1st layer-line
10
9
8
7
Effective lattice
volume/molecular volume
0.96
1.07
1.20
1.38
Table 2. Molecular volume estimation for N M V
Residue
Ala
Val
lie
Gly
Leu
Pbe
Pro
Met
Trp
Ser
Thr
Cys
Asp
Tyr
Glu
Lys
His
Arg
Volume*
(nm 3 X 10 3)
91.5
141.7
168.8
66.4
167.9
203.4
129.3
170.8
237.6
99.1
122-1
105-6
124.5
203.6
155.1
171.3
167.3
173.5
Number of
Volume
residues~"
(nm 3)
32
2-928
13
1.842
8
1.350
13
0.863
21
3.526
9
1.831
20
2.586
3
0.512
3
0-713
15
1.487
13
1.587
2
0-211
24
2.988
6
1.222
18
2.792
10
1-713
2
0.335
10
1.735
Total = 30.221 nm 3
Vol. of 5 nucleotides = 1.50 nm 3
Total vol. (protein + nucleates) = 31-721 nm 3
* From Chothia (1975), except for Arg which is from Zamyatnin (1972).
t Short (1982).
arrangement with a = 10.6 + 0.1 nm, with a helix pitch of 3.30 _+ 0.03 nm (Tollin et al., 1967).
Using the amino acid volumes given by Chothia (1975) and Z a m y a t n i n (1972), the total volume
of a protein subunit can be determined (Table 2). The R N A content implies that there are
probably five nucleotides associated with each protein subunit (Bancroft et al., 1980) and the
volume of these can be estimated from the results of X-ray crystallographic studies of
nucleotides. Thus, the total volume of protein plus nucleotides can be estimated.
The volume of the dry hexagonal cell of height 3.30 nm is 10.6 x 10.6 x V~/2 x 3.3 =
321.1 nm 3. Taking into account the axial hole in the virus particle, which studies of tulip virus X
(Radwan et al., 1981) and barrel cactus virus (Richardson et al., 1984), also potexviruses, suggest
is of radius about 1.5 nm, the effective volume available for the protein and R N A is 297.8 nm 3.
Table 1 lists the ratio of the effective lattice volume to the molecular volume for q values in the
range 7 to 10. These may be compared with the ratio value o f 1.08 for T M V (Makowski &
Caspar, 1978) and 1.09, 1.20 and 1.11 for bacteriophage Pfl, fd and X f respectively ( N a v e et al.,
1981) where the number of subunits per helix turn are known from studies of heavy-atom
derivatives. It thus seems that q values of 8 or 9 would be acceptable for N M V .
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O f t h e t w o p o s s i b l e values o f q w h i c h are o b t a i n e d f r o m t h e F o u r i e r t r a n s f o r m c a l c u l a t i o n s
a n d t h e m o l e c u l a r v o l u m e c a l c u l a t i o n s , o n l y q = 9 is c o n s i s t e n t w i t h b o t h . W e c o n c l u d e ,
t h e r e f o r e , t h a t t h i s is t h e t r u e v a l u e o f q for N M V .
A v a l u e o f q = 9 c o r r e s p o n d s to 44 s u b u n i t s in 5 t u r n s o f t h e helix, or 8-8 s u b u n i t s p e r t u r n .
T h i s is in a g r e e m e n t w i t h t h e h y p o t h e s i s o f R i c h a r d s o n et at. (1981) t h a t t h e n u m b e r o f p r o t e i n
s u b u n i t s p e r t u r n o f t h e helix i n t h e p o t e x v i r u s e s is close to, b u t slightly less t h a n 9, a n d t h a t
d i f f e r e n t viruses in t h e g r o u p differ only in t h e f r a c t i o n a l d e p a r t u r e f r o m 9.
We wish to thank Dr M. N. Short for sending us details of the amino acid composition.
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