4.15 Ciclo de minado inicial
4.15.3 Limpieza y extracción del material disparado
1.3.1 X-Rav Crystallographv.
T h e m a jo r ity o f k n o w n p r o te in s t r u c tu r e s h a v e b een
d eterm in ed by X-ray crystallography (Blundell and Jo hnson, 1976).
The first stage of crystallography is obtaining a well-ordered protein
c ry stal. P ro te in cry stals are grown from su p e rsa tu ra ted solutions.
Proteins do not readily form regular crystals; they are generally an
e llip so id a l shape and do not p ack into space fillin g geom etries.
with neighbouring molecules in a few, small regions. The large gaps
in the protein crystal are filled with solvent molecules. Although the
large amount of disordered solvent present (often 50% of the crystal)
makes it difficult to obtain w ell-ordered crystals, it does mean that
the p ro tein ad o p ts a c o n fo rm a tio n in the cry stal w h ich is not
markedly d ifferent from that adopted in solution.
O n ce a c ry s ta l has been o b ta in e d it is ir r a d ia te d w ith
m onochrom atic X-rays. M ost X-rays pass straight through the crystal,
b u t som e i n te r a c t w ith a to m s in the p r o te in m o le c u le . T his
in te rac tio n c au ses X -ray s to be scattered in all d ire c tio n s. The
scattered X-rays interfere with each other, generally cancelling each
other out. However, in certain positions, and for certain angles, the X-
rays interfere constructively and a diffracted X-ray beam leaves the
crystal. Such beams are recorded as a spot on a photographic film or
e le c tro n ic d e te c to r, p lac ed a k n o w n d ista n c e from the cry stal.
Recording the amplitude of the diffracted beam loses a vital piece of
inform ation, the phase of the beam s which leave the crystal. This
pro b lem is c ircu m v en ted by intro d u cin g a small n u m b er o f extra
atom s into the pro tein cry stal. The atom s w hich are added are
’heavy', m etal atoms. These atoms make a large contribution to the
X -ray p a tte rn , and so only a few ato m s need be ad d ed , thus
preserv in g the protein structure. By e x am in in g the c h an g e in the
diffraction pattern it is p o ssib le to determ in e the p o sitio n of the
m etal atoms, and their phases. Use of two heavy metal derivatives
enables the phase contributions from the protein to be estimated.
Knowledge of both the amplitude and the phase of each spot of
the X-ray diffraction pattern allow s an electron density m ap to be
calculated. This map is imperfect. Errors arise due to disorder in the
crystal, incorrect phase determ ination and inaccuracy from the X-ray
d iffra c tio n e x p e rim e n t itself. The ele ctro n d e n sity m ap is then
interpreted. This involves tracing the path of the protein m ain-chain
through the electron density, and positioning side-chains so that they
a g re e w ith th e o b s e rv e d d e n s ity as w ell as p o s s ib le . T h is
in te rp retatio n is often su b jectiv e, and is m ade m ore d iffic u lt by
portions o f the map which contain little or no observed density, a
particu lar pro b lem at the protein surface, where the side-chains are
m ost mobile. The amino acid sequence of the protein is essential for
the correct tracing of the protein backbone.
The next step is to refine the interpreted structure. This is a
c o m p u te riz e d p ro c e d u re to m in im ise the d iffe re n c e b e tw ee n the
o b s e r v e d d i f f r a c tio n a m p litu d e s and th o se c a lc u la te d fo r the
interpreted structure. This is done by m aking small changes in the
p o sitio n o f ato m s, and is often lin k ed to a p ro c e d u re w hich
norm alises the bond lengths and bond angles seen in the protein.
During the refinem ent process it is often possible to assign a B value
to each atom. This value indicates the relative mobility of that atom
( A r t y m iu k , et al., 1979).
It is impossible to state the error in the atomic co-ordinates of
a protein which has been determ ined by X-ray crystallography. Two
m easures o f quality that are often quoted are the resolution o f the
structure and its R-factor. The resolution is simply a m easure of the
a m o u n t of diffraction data collected. M ore ob serv atio n s m eans the
electron density map is more detailed, and hence the final structure
will be better. Resolution is quoted in Angstroms, a high value for the
resolution im plies low quality. A resolution of 5Â allow s only the
general shape of the protein to be established, possibly with some
se co n d a ry stru c tu re assig n m en t. R e so lu tio n s o f aro u n d 3Â allow
sid e -c h a in p o sitio n to be d e te rm in e d , m any larg e p ro te in s are
determined at this level. Resolutions of between 1Â and 3Â produce
very well defined electron maps, with spheres of electron density at
atom positions. Small proteins are often resolved at this level. The
resolution has several draw backs as a m easure of quality. It is a
global m easure and hence incorrect areas of the structure cannot be
highlighted and it is a m easure of the quality of the electron density
map. In co rrect interpretation can still result in in co rrect structures.
A better m easure, which overcom es this second problem is the R-
factor. The refin em en t process attem pts to m inim ise the difference
betw een observed and calculated diffraction intensities, the R -factor
is a p ercen tag e disagreem ent between the two sets o f data. A R-
fac to r o f 0% im p lies th at all the o b se rv ed d ata is c o m p lete ly e x p la in e d by the p ro tein stru c tu re o b tain ed . R a n d o m a g re em e n t
produces a R-factor of roughly 60%. Most proteins have an R-factor
of b etw een 10% and 20% , after refin e m e n t. In m in im is in g the
difference betw een observed and expected diffraction intensities the
refinem ent stage is effectively m inim ising the R factor. This could
lead to a good R-factor for a structure that is incorrect. It has been
suggested that some of the diffraction data be w ithheld from the
refinem ent procedure and that this data be used to calculate the R-
factor (Bruanger, 1992).
1.3.2 N uclear Magnetic Resonance.
The other m ethod that has been used to d e te rm in e precise
three d im en sio n al protein structures is n u clear m agnetic resonance
sp e c tro sc o p y (N .M .R .) (W u t# rich , 1990). T his m eth o d has only
recently been extended to proteins from small m olecule work. Over
th irty p ro tein stru ctu re have been so lv ed , m o stly w ith sizes of
around 50 residues, but larger structures such as Interleukin 8 with
144 residues have been solved (Clore, et al.y 1990).
N .M .R . relies on the m ag n e tic m o m e n ts (o r spin) o f the
h y d ro g en nucleus (i.e. a p roton). W hen p roteins are p laced in a
strong m agnetic field the spins of the hydrogen nuclei tend to align
p arallel or antiparallel to the im posed field. It is then p ossible to
e x c ite th e sp in s by s u b je c tin g th e p r o t e i n to a p u ls e o f
electrom agnetic radiation. This excitation will only occur at a certain
frequency, which will be in the same broad area of the spectrum for
all protons but which also depends upon the local environm ent of
th e p ro to n . T h e v a ria tio n o f c h a r a c te r is tic fre q u e n c y d ue to
environm ent is called the chemical shift (Figure 1.6a). Conventional
one dim ensional N .M .R ., as used for small m olecules, m easures the
absorption spectra of the sam ple, in the relev an t freq u en cy range.
A lthough this, in principle, gives a unique signal from each n o n
identical hydrogen, the absorption peaks overlap. This is because the
d ifference in chem ical shift is sm aller than the resolving pow er of
the experim ent. To overcom e this problem two dim ensional N.M .R.
was introduced. Here the experim ent m easures interactions betw een
tw o se p a ra te spins at d if f e r e n t fre q u e n c ie s, co i and o) 2. T h ese
interactions are represented as cross-peaks on a 2-D N.M .R. map, as
shown in Fig u re 1.6b. The exact nature o f the N.M .R. experim ent
d e te r m in e s the ty p e o f sp in -sp in in te r a c tio n e x a m in e d . C O SY
( c o r r e la t io n s p e c t r o s c o p y ) sh o w s h y d r o g e n a to m s w h ic h are
connected by three or few er covalent bonds. H ence C O SY spectra
show hydrogen atoms which belong to the same residue. The NOESY
( n u c l e a r O v e r h a u s e r e f f e c t s p e c t r o s c o p y ) e x p e r i m e n t sh o w s
in te rac tio n betw een h y d rogen atom s th at are less than 5Â apart.
(a) ia) ic) (b) CH3- C H2 - O H W) (0 %
I
6 5 4 3 Chemical shift (ppm) (b ) 0 00 1 9 8 7 6 5 4 3 2 F i g u r e 1.6 c h e m i c a l shift ippm)(a) A 1-D ^H-NMR spectrum for ethanol. The chemical shift for each hydrogen atom in the labelled groups is clearly visible. The signal from the CH3 group hydrogens is split into three peaks and the
signal from the CH2 group hydrogens is split into four peaks, due to
experim ental conditions.
(b) A 2-D NOE NMR spectrum of the C-terminal domain of cellulase. The off-diagonal peaks represent interactions between hydrogen atoms that are separated by less than 5Â in space.
Both diagrams are taken from Branden & Tooze (1991) 35
Hence N O ESY spectra give inform ation on which residues are close
together in the folded protein, despite being rem ote in sequence.
To solve a protein structure by N.M.R. the 2-D spectra must be
correctly interpreted to give a series of distance constraints between
atom s. If sufficient constraints are obtained the relative positions of
all the atom s can be calculated. In real cases certain areas of the
protein structure are insufficiently constrained, this leads to multiple
possibilities for the solution and hence a family o f possible structures
are obtained. The quality o f an N.M .R. structure cannot be assessed
by the sam e m easures as a c ry stallo g rap h ic structure. Instead the
average root mean square deviation of each structure in the solution
f a m i l y fr o m a r e p r e s e n t a t i v e s t r u c tu r e is c a l c u l a t e d . T h e
rep resen tativ e structure is the average for all the possible solutions
found, this structure has no physical m eaning and is not likely to be
a m em ber of the solution family.
C om parisons betw een independently solved X -ray and N.M .R.
stru c tu re s fo r the sam e pro tein have been m ade (B ille te r, et a l ,
1989). Such studies show a large degree of agreement of between the
different m ethods, particularly in the interior of the protein. At the
exterior of the protein, where crystal structure tend to have large B-
valu es and N .M .R . structures have few c o n strain ts, d ifferen c e s in
side-chain orientation are found.