, n 1997.

1
?????????? ????????? ?????? ? ????? ????????
???????? ????????? ????? 5 ????? ???????? ??????
? ??????????? ?????? ?????????????? ??????? ???.
????????????? ?????? ?? ???????????? ????? ,
??????????? ? ????? ???????? ???????? (n? ???????
1997). ??????? ???? ??????????, ??? ???????????
???????? ????? ? ????????? 1-25 kDa , ?
?????????? ? ?????? 8 kDa ? ?????? ?????????
???????? ????? ???????????? ?????? ?????? 25
kDa. ???? ????????? ????????????? ?? ????????
?????????, ??????? ?????????? ??????????? ?????
??? ??? ??????????? ?????? ? ???????????? ??????,
?????? 30 kDa. ??? ??????????? ???????????
?????? ????? ??? ?????????? ??? ???????
?????????, ??? ??? ??????????? ??????.
2
????? ?????????? ??????? (?? 10 ???)
?????????? ????????? ??????????? ?????????? ??
?????? ????)
The completion of the first protein NMR
structure brought new, unexpected challenges.
When I presented the structure of BUSI (Fig. la)
in some lectures in the spring of 1984, the
reaction was one of disbelief, and because of the
dose coincidence (Fig. Ib) with results from an
independent crystallographic study of the
homologous protein PSTI (porcine pancreatic
secretory trypsin inhibitor). It was suggested
that our structure must have been modeled after
this crystal structure. In a discussion
following a seminar in Munich on May 14, 1984,
Robert Huber (Nobel Prize in Chemistry, 1988)
proposed that we settle the matter by
independently solving a new protein structure by
X-ray crystallography and by NMR. For this
purpose, each one of us received an ample supply
of the a-amylase inhibitor tendamistat from
scientists at the Hoechst company. Virtually
identical three-dimensional structures of
tendamistat were obtained in our laboratory by
NMR in solution and in Robert Huber's laboratory
by X-ray diffraction in single crystals. The
refined tendamistat structure was published in
Journal of Molecular Biology as a 50-page report,
and the addendum to that paper dearly illustrated
the impact of structure determination by NMR.
Wuthrich K., (2001) The way to NMR structure,
Nat. Struct. Biol. 8, 923-925
3
????????? ??????????? ???????????? ?? ??? ??????
(?? ?? ????????, ? ?? ????????!!!)
1. ?????????? ? ???????? ??????????? (lt5Å)
Nuclear Overhauser Enhanced Spectroscopy ????????
???????? ?????????????? ?????????? ? ????????
??? ?????????? ?? ??????? ??????????? (N??).
??? ??? ???? ?????? ??????? ?????????????? ??????
??????? ?????????? ????? ?????????, ?? ???
????????????? ????? ?????? ???????? ???
?????????? ?????????? ????? ????. ?????? ????????
?? ??? ?????? ??????????? ?????????? ???????
???? ?????????? ??? ??????????? ??????????
????????? ????????????? ????????????. ??
??????????? ???????? ????????????, ?????
????????? ?? ???????? ???????? ?????? ?????????
?? ????.

?
2. ?????????? ? ??????? ??????????? (gt5Å)
???????????????? ?????????? ???????????? ?
????????????? ???????? ( phospholipid bicelles),
??????????????? ????? Pf1 ? ?.?.
4
????? ???????? ? ???????? ??????? (2O-60 kDa) 
1) ??? ?????? ? ???????????? ??????, ?????? ???
10 ??? ??????????? ???????, ?????????? ?? ???????
1H 2D ???????? ?? ??????? ??-?? ???????
??????????????? ????? ????????. 2) ??? ?????? ?
???????????? ?????? ? ????????? 15-20 kDa
??????????? ???????????? (???????)????????,
?????????? ?? ?????????? ????? ?????? 1H, 13C ?
15N  2)  3) ??? ??????, ? ????????????
?????? ?????? ??? 20 ?D? ??? ????????? ????????
?????????? ?????????????? ????????, ???????
???????? ??????? ?? ???????????? ?????. ???,
??????? ??????????? 3D ????????? ????? ?
???????????? ?????? 30 ?D? ?????????? 90
??????????????. 1)  4) ??? ??????, ?
???????????? ?????? ?????? ??? 30 ?D? ???????????
?????????? ????? ???????????? ??????? ?? 13Ca and
13Cß ??????. ??????????? ??????? ???????
?????????????? (95) ?????????? ? ????????
???????? ?????????. ???????????? ???????????
????? ??????? ?????????? ????? (TROSY).
???? ????????????!! ??????????? ?????? ???.
5
????????? ??????? ?????? Goto and Kay (2000),
Current Opinion in Structural Biology, 10,
585-592.
?????????????? 1) ?????????? (?????? ???
?????????) 2) ??????????? (?????????? 2H
???????????) 3) ??????????? (??????? ?????????
????? ???????, ??????, ???????, ?????????? ???
????????????? ?????????????? ????????  4)
????????????? ??????? (???????? 1H ? 2H
????????????? ????? ? ??????? ????? ???????)
13C ? 15N ??????? 1) ??????????? ?. Coli ??
????????????? ??????, ?????????? ???? ??????? 13C
? 15N. 2) ????????????? ??? ???? ???? Pichia
pastoris (?????? ?????????? ???????).  3)
????????????? ???????????? ?????? ??????????.
????????? ??????? ??? Tolbert and Williamson
(1996) J. Am. Chem. Soc, 118, 7929-7940
?????????????? 1. ????????????? ?7 ??? ??????????
??? ???????? ???????? ??? ????? 2. ??????????
??????????? ??????? ??????????? ? ???????????????
??????? ??????????????? ??????????? 3.
??????????? ??????? H5' ???????? ? ???????? ? H3'
? ?????????. 4. ?????????? ?????? ??????????? ??
???????????? ??????? ?????? ? ?????????
13C ? 15N ??????? (??? ?? ??? ? ??????)
????????? ??????? ??? (????? ????????????0 Gardne
r and Kay (1998) Annu. Rev. Biophys. Biomol.
Struct,. 27, 357-406
6
??????? ? ????? ??????? ?????????????????
?????????. ? ????????? ????? ?????????, ???
????????????????? ????????? ? ???????????? ??????
?????? 60 ?D? ?????????? ?????? ???. ??????, ?
??????? ????????? ????????? ????? ???????????
????? ?????????.
?? ??????? (??????) ???????? ????????? ?????
GroES (72 kDa). ???? ????? ???????? ???????????
??????, ????????? ?? 7 ??????????, ???? ??
??????? ???????? ??????? ??????. ?? ???? ??
??????? ????? ???????? ?????? ????????? GroES ?
GroEL (472 kDa). ??????? ?????? ???????? ?????
GroEL, ??????? ?? ?????? ???????????? ? ?? ?????
? ??? ????????????. ??? ???? ??????????? ????????
????????? GroES ? ????????????? ????????? ??
??????? ?????????.
7
??? ? ????? ??????????? ????????
??????? ???? ??????????????????? ???????? ??
????? ?????? ??????????? ?????????. ???????????
NMR-ONLY ????????, ??? ?????? ????? ???
????????????? ??? ??????????? ?????????, ?????
??? ??????????? NMR_XRAY ????????, ??? ? ??? ?
????????????? ????????? ???? ????????????.
??? ????????? ??????????????? ????? ?????? ?????.
? ???????? ?????, ?????????????? ?????? ????
??????? ? 126 ?? 226 ???????, ????? ???
????????? ????? ???????? ? 23 ?? 126 ??????? ??
??????????????? ?????.
??????? ??????? ??????????? ??????? ????????
??????????????? ????? (BFP) ? ??????????? (B) ?
?????????? Ca2 (D). ??? ???????? ?????????
????????????, ??? ? ??????????? Ca2 ????
??????? ????????? ??????? ?????????, ????? ???
??? Ca2 ????? ?????? ??????????? ???. ????????
????????, ??? ????????? ?????? ???????????
??????? ??????? ????? 15 min
8
??? ??????????? ??????
?????????? ?? ????????? 5 ???, ??????????
???????????? ???? ????? ???????? ???????????
??????, ?????????????? ??????? ???. ??? ???? ?
1997 ????, ???? ???????????? 137 ????????
??????????? ?????? ? 784 ???????? ????????, ?? ?
2001, ???? ??? ???? 419 ???????? ???????????
?????? ? 1775 ???????? ????????. ?????
??????????? (14) ??????????? ? ???????.
?????????? ????????? ? ????????? ?-???,
??????????? ??????? ???. ? ????????????? ????
?????? ???????? ???????????? ?????? ??
????????????? ?????????.
??????? ????????????? ???????? ????????? ?
??????? ???????????? ????? ??? ??? ???????????
??????????? ??????. ???, ???? ??? ?????? NOE
???????? ??????? ???????? ????? ?????????,
???????????? ?????? ?? ???? (?), ?? ???
??????????? ?????? (?) ????? ???????? ??????????
?????????, ???????????? ?????? ?? ????. ???
?????? ???????????? ??????? ?????? ?????
??????????? ??????, ????????? ?????? ???
9
??? ? ????????. ???? ?????? ???????????
?????????????
???-??????????
??????????? ?????????? ????????????? ???????
????????? ????? ?????????. ??? ???? ???????????
?????? ??????????? ?????????? ?????????????
(????? ????? 1-10Å), ?????????????? (????? ?????
????? 0.1-1 ??) ? ???-??? (????? ????? gt 1 ?).
????? ????? ????? ????????? ???????? ???????????
????? ??? ?????. ????? ??????? ? ?????????????
????????? ? ????????? ????.
??????????????
???? ????????? ???? ????? ???????? 1 x 10-5
?????/c?, ?? ???????, ????????? ???? ?? ????? ??
1 ?? ????? ????? ??????? ? ??????????? ??????? ?
425 Hz. ??????? ??????? ??????? ??? ? ????? ?????
????? ?????????????? ?????? ? ????? 1 ?? ?????
?????????? ?????????. ??? ????????? ???????????
??????????? ??????? ?????????? ????? ???
????????? ?????????? ???? ?? ???????
???????????????? ????????????. ????? ??????
?????????? ??????????.
10

Magnetic resonance image of Homo sapience. The
image took approximate 5 min to record. Bar 10 mm
Light microscopy (a) and microscopic resonance
imaging (side view, (b) and top view (c) of a
living embryo