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Resonancia Paramagnética Electrónica

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Elsevier

BBM 00710

correlation times and separation of rotational and

polarity effects from EPR spectra of spin-labeled

biomolecules in a wide correlation time range

Heinz-Jiirgen Steinhoff

lnstitut ]'fir Biophysik, Ruhr-Universitiit Bochum, 4630 Bochurn, F,R. G.

(Accepted 15 August 1988)

Summary

A method using nitroxide radical spin labels for determining both the isotropic rotational correlation

time ZR and the environmental polarity of the label is described. By means of a least square fitting

method, the values of an effective hyperfine tensor A' and of an effective g value tensor g ' of randomly

oriented spin labels are determined from X-band EPR spectra on the basis of an effective time-indepen-

dent Hamihordan. The traces of the tensors deliver the information about the environmental polarity of

the label and are not dependent on the rotational correlation time "rR. A new averaging parameter S

(~'R), calculated on the basis of the principal values of the tensor A', permits the evaluation of the

rotational correlation time z R in a very wide time range between 1 0 10 and 1 0 6 s. An application of

this method to spin-labeled methemoglobin over a large temperature range and in environments of

different polarity is discussed.

Introduction

The sensitivity of spin-label spectra to the rate of nitroxide rotational motion has

long been known [1,2]. Therefore, if a spin label can rigidly bind to a macromolc-

cule, its electronic paramagnetic resonance (EPR) spectrum should directly reflect

Correspondence address: H.-J. Steinhoff, Institut fi.ir Biophysik, Ruhr-Universit~it Bochum, 4630 Bochum.

F.R.G.

238

the motion of the macromolecule itself or some residual motion (librational motion)

of the label with respect to the whole macromolecule [3-5]. Rotational correlation

times ri~ for isotropic motions of slow-tumbling spin labels or spin-labeled biopoly-

mers have been determined by use of appro:dmate methods as we11 as simulation

methods [2]. These approaches utilize the property' that the positions of the outer

peaks in the X-band spin-label spectra (their separation is approximately 2Azz,)

shift inward from their rigid limit position, 2Azz, if the rotational motion increases.

On the other hand, the principal values of the hyperfine tensor __Aand of the g value

tensor g of a nitroxide radical depend significar~tly on the polarity of its environ-

ment [6,7]. Since a protein possesses hydrophobic and hydrophilic regions, it must

be considered that the environment of a protein-bound spin label may change its

polarity during a temperature-induced conformational change of the protein.

Another explanation of an observed temperature dependence of the peak separation

in the spectrum ma t' be the N O ' - H X hydrogen bound formation by, the spin label

[8,9~.

A special problem has been to separate the spectral effects of nitroxide libra-

tional motion from those due to hydrogen bonding interactions or polarity' change.

This problem may' bc partially overcome if one considers not only the hypcrfine

separation 2Azz but also line-width values of the absorption spectra [4,5,13]. In the

present work it is dcmonstra:cd, that this problem of" separation may be especially

overcome through determination of thc complete tensors g and A of the spin-labeled

protein using a fast least square fitting method. In the case of polarity change of the

spin-label environment, the change of the hyperfine separation 2Azz is correlated to

the change of the hyperfine tensor (TrAy trace. On the other hand. if an observed

change of the hyperfine separation is due to librational motion of the labcl, the trace

of the tensor _A must net alter, which follows directly from the rotational invarianee

of the trace of a tensor [111.

Therefore the starting point of the separation method presented here must be the

determination of the tensors g and A also in the slow motional region. However, a

least square fitting method in the slow tumbling region (~R -- 10 7 S) on the basis of

Freeds prograln [2] is very time consuming. A ~ypicat fit with seven parameters

including ten steps will last up to 2300 s (depending on rR) on a Cyber 855,

delivering the tensors g and A and the rotational correlation time ~'R- Instead of

this, the method presented here describes the EPR spectra of tumbling spin labels

using an effective time-independent Hamiltonian. This is a drastic simplification

and its relevance to experiment and theory has to be checked very carefully. Some

points of practical interest arc to be mentioned. To ensure the uniqueness of the fits

the number of parameters is reduced to a minimum: the six principal values of the g

and _A tensor. The line width function is determined from the spectrum in an

independent step. The fitting pr(rcedure is very fast so that an implementation of the

method using a personal computer is possible.

As an example of an application of this method, the tensors g and A of the spin

1abel .. :-(1-oxvl-_. 6.6-tetramethvl-4-piperidinvl) iodoacetami-de (JAA6) bound

within methemoglobin in different environments are determined in a temperature

range between 80 and 330 K.

239

Materials

Oxyhemoglobin was prepared from fresh horse blood samples by the method of

Benesch et al. [12]. Spin labeling (JAA6) followed the procedure of McConnell et al.

[13]. The oxidation of the labeled oxyhemoglobin to methemoglobin was achieved

by addition of a threefold amount of K 3 (Fe(CN)~,). The sample was desalted by

running it through a column of Sephadex G-25. Polycrystalline samples were

prepared by mixing the solutions of labeled methb with buffered anamonium sulfate

according to the method of Perutz [14].

EPR spectra were measured on home-made X-band and K-band (22 G H z )

spectrometers equipped with a modified Oxford ESR 9 variable temperature acces-

sory. The microwave power used was 0.1 mW, the modulation frequency was 50

kHz and the modulation amplitude 0.4 10 - 4 T. The magnetic field was measured

with an A E G Telefunken N M R instrumentation. 2,2-Diphenyl-l-picrylhydrazyl

(DPPH) powder served as a g value standard ( g = 2.0037). After analog-digital

conversion, the spectra were recorded in a personal computer (CBM 8296, Com-

modore) and then transmitted to a Cyber 855 (Control Data). There the tensors g

and _A were determined as described in the following section.

Methods

samples

The EPR spectra of a nitroxide radical with spin 1 / 2 having fixed orientations

can be accounted for with the basic spin Hamiltonian

where H is the external field vector, /~ the Bohr magneton, S the electron spin

operator, I the nuclear spin operator, g the electron g value tensor and /t the

electron nuclear hyperfine interaction tensor. To a good approximation [15], the

eigen values of the Hamiltonian Eq. (1) are given by

E = g ( O , ~ ) B e H M s + A ( O , c ~ ) M s M I, (2)

with

g(O,O) = g x x l z x + g v v l z y + g z z l z z ,

where gxx, ,g'YY, gzz and Axx, A y y , Azz are the principal values of the tensors g

and A, respectively, a n d / z x , lzv, /zz are the squared direction cosines between the

nitroxide x, y, z axes and the laboratory magnetic field direction (z): /zx = ( - s i n 0

240

COS~) 2, /ZY = (sin0, sin~) 2, lzz = (cos0) 2- Ms and M l are the eigen values of Sz

and lz, respectively. Considering the selection rules Eq. (2) gives the resonance

frequencies of every" spin-label orientation in the external magnetic field. A F o r t r a n

program was written to calculate the corresponding E P R spectrum. The E P R line

positions are calculated in steps of 3 for 0 and q~. The intensity of each EPR line

of a r a n d o m distribution of spin-label orientations in the laboratory fixed coordi-

nate system is proportional so the n u m b e r of molecules d N, for which Eq. (3) holds:

After summing the stick spectra within a 0.5 G range of H, the line shape is applied.

The line shape function may be evaluated from the first derivative of the experimen-

tal EPR absorption spectrum. In the case that the difference between the principal

values Azz and Axx, Ay~- is large c o m p a r e d to the line width A (a condition,

which is fulfilled in the case of nitroxide labels), the resonance lines of the first

derivative of the EPR spectrum at the positions H+ = (hv++_ A , c z ) / ( g z z B ~ ) are

identical with the line shapes of a single resonance tine [16]. Here we suppose that

the line width of the resonance lines does not depend strongly on M~ or g. This

assumption will be proved experimentally in the next section. The convolution of

the calculated stick spectrum with the first derivative of the line shape determined

from the experimental E P R spectrum yields the desired theoretical E P R spectrum f,

which is now a function of the six principal values of the tensors g and _A only. By

means of the subroutine S Q U F I T [17] the initially chosen values of g and A are

varied iteratively until a m i n i m u m of the chi-squared value Q is ob]ained (least

square fitting). Alternatively the determination of the line shape function m a y be

included into the fitting, increasing the n u m b e r of parameters from six to nine. A

complete fit including ten steps takes less than 10 s on a Cyber 855 (about 30 min

on a personal computer without floating point coprocessor).

The next step is to prove the applicability of the fitting method to experimental

spectra. The immobilization of the labeled methemoglobin molecules was achieved

by crystallization in a m m o n i u m sulfate solution, by freezing of a methemoglobin

solution or by lyophilization. As an example Fig. 1 shows the calculated and the

TABLE 1

VALUES OF THE TENSORS g' AND A', DETERMINED IN THE X- AND K-BAND AT T = 180 K

X-band K-band

(u = 9.00 GHz) (u = 22.00 GHz)

gxx 2.0089 + 2 x 10 -4 2.0089 + 5 x 10-4

gyy 2.0064+210 4 2.0060+510-4

gzz 2.0021 + 2 x 10-4 2.0021 + 5 x 10-4

Axx/MHz 17.4 +_1.2 18.2 +2.0

Ayy/MHz 21.3 *1.2 18.6 +2.0

Az~/MHz 104.5 _+0.1 104.0 + 2.0

1/3 TrA/MHz 47.7 _+0.4 46.9 +_2.0

241

(3

I I I

B/Testa

I I I I

0.810 0.815 0.820 0.825

B/Testa

Fig. 1. EPR spectra of polycryst~fine methemoglobin-JAA6 at T=180 K. (a) t,o=9.0 GHz: (b)

% = 22.0 GHz. The dotted lines show the least square fittings to the experimental spectra.

Excellent agreement is o b t a i n e d between the calculated a n d the e x p e r i m e n t a l curves.

The tensors g a n d _A d e t e r m i n e d from both frequency b a n d s are shown in T a b l e l.

2a2

The corresponding values coincide. The greater standard deviation of the values

determined from the K-band compared to those determined from the X-band is due

to a lower signal to noise ratio. This result shows that the approximation in Eq. (2)

is valid in the K-band too.

No significant change in the values of g, _A and Q was observed when we

extracted the line shape function from the h~-gh field peak instead of the low field

peak. So the dependence of the line width on M~ and g is small and the assumption

of a single line shape function for the whole EPR spectrum is justified.

Because of the choice of a time-independent Hamiltonian Eq. (1) the introduced

method of determining the tensors can firstly be applied to strong immobilized

samples. From the work of Freed [2] it is known that the hyperfine separation and

the intrinsic line width of the first derivative of the absorption spectra is insensitive

to molecular motion, if the correlation time r is greater than 10 -7 s. Motional

fluctuations with corrdation times less than 10--7 s will produce partial averaging of

the tensor dements. We describe the spectrum in this time range introducing

effective g ' and A' tensors and this yields an effective time independent Hamiho-

nian H~ff. The questions now to be answered concern the properties and appLica-

tions of this effective time-independent Hamiltonian. A first consistency check is

that one must have TrA = TrA', which follows directly from the rotational invari-

ance of the trace of a tensor. We have calculated EPR spectra which are char-

acterized by motional fluctuations with correlation times between 6 l 0 - 7 and

10 10 s using the stochastic Liouville method of Freed published in [2] with

assumed values of the tensors g and A, which we shall call 'true' in the following.

These spectra were fitted with the method described in the preceeding section

leading to values of the effective tensors g ' and A', which depend on the values of

%. The relative depilation of TrA' from the ' true' value TrA, o.r~A, and the deviation

of Trg' from Trg, o.r~~, are shown in Fig. 2. In the time range of % above

0.3 ]0 -v s and below .10 -9 s the values of TrA' of the best fits deviate less than !%

from the given value of TrA, the deviation in Trg' is less than 5 10-5 in this

range. Even in the critical correlation time range between 10 -~ and 10 .-9 s the

systematic deviations in TrA' and Trg' are less than 3% and 10 -4, respectively. The

fluctuations of ~r~g are mainly due to the finite step width of the spin-label

orientations for 0 and q). While the traces of the fitted tensors are almost

independent of the correlation time %, their principal vahies change from the rigid

limit values to the complete averaged values Axx =A~. v = A z z = 1 / 3 TrA and

gxx = gw" = gzz = 1 / 3 Trg in the rapid motion limit: the value of AT~z changes

from !05 MHz to 47.6 MHz for example. The independency of TrA' on % is an

essential result and will be used to characterize motional effects on EPR spectra and

to separate other effects. We introduce an averaging parameter S characterizing the

degree of averaging on the basis of the tensor A:

Sz = A z z - 1/3 TrA'

Azz - 1 / 3 YrA ' (5)

243

2

~'T. A.]0

/.

~.rg"10

-1 i i i i i i . i l i L , ,, i , ,L,~i[ i,.

Fig. 2. The de,Aation of TrA' and Trg' from the 'true' traces of the tensors, TrA and Trg, respectively, as

a function of the correlation time ~'R for Brownian rotational diffusion. The deviations o are defined as

OTrA = (TrA' - T r A ) / T r A (X) and ol-rg= (Trg' - T r g ) / T r g (C). The curves were determined from

fittings of spectra calculated on the basis of a time-independent effective Hamiltonian to spectral

simulations using the exact stochastic Liouville method of Freed [2]. The parameters for the calculated

spectra in the notation of [2] are: L_<I6, K_<12, A x x = 7 . 4 G, Avv=6.1 G, AT,z=37.5 G,

gxx = 2.0089, gvv = 2.0063, gzz = 2.0023 and the peak-peak first derivative line width is 3.0 G.

or, i n c l u d i n g all p r i n c i p a l v a l u e s

i = X,V,Z

S = " (6)

]A,- 1/3 Tra]

i = x,y,z

1 for the rigid l i m i t case, A~t = A , , to z e r o in the c a s e o f c o m p l e t e a v e r a g i n g . T h e

v a r i a t i o n o f S w i t h ~R is s h o w n in Fig. 3 for the B r o w n i a n d i f f u s i o n m o d e l . It

s h o u l d be n o t e d t h a t S is d e f i n e d in the w h o l e r a n g e o f ~'R f r o m 10 - 6 to 10 -10 s. So

t h e r a n g e o f ~R, w h i c h c a n b e e v a l u a t e d f r o m an e x p e r i m e n t a l s p e c t r u m u s i n g a

r e l a t i v e l y s i m p l e a n d fast m e t h o d , c a n b e e x t e n d e d to c o r r e l a t i o n t i m e s less t h a n

7 x 10 - 9 s, w h i c h h a d b e e n the l o w e r l i m i t for the s i m p l i f i e d m e t h o d g i v e n in [2].

T h e r e a s o n for t h a t l i m i t a t i o n is t h a t the o u t e r lines b e g i n to c o n v e r g e to the

m o t i o n a l l y n a r r o w e d s p e c t r u m a n d the p a r a m e t e r S d e f i n e d in [2] b e c o m e s u n d e -

f i n a b l e . C o m p a r e d to this, Fig. 3 c a n b e u s e d as an e m p i r i c a l c a l i b r a t i o n c u r v e for

244

0-9

08

07

06

0.5

0.4

0.3

0"2f

ol

Fig. 3. The parameters S z (o) and S (D) versus rotation',d correlation time. The curves were determined

from fittings of spectra on the basis of a zime-independent effective Hamihonian to spectral simulations

using the stochastic Liouville method. The parameters for the simulations are the same as in Fig. 2.

determining r R values from the effective hyperfine tensor ,4' in the whole time

range from 10 -6 to ]0 -1 s.

The values of the parameters S and TrA' shown in Figs. 2 and 3 were calculated

starting the fitting with varying initial sets of g ' and A'. The fitting converged in all

cases if the initial choice of g ' and A ' corres-ponds to S values, which deviate less

than 0.3 from the final (' true-~) value of S. The initial values of TrA' and T r g ' m a y

deviate from their true values more than 20% and 2 10 -3, respectively.

EPR spectrum of spin-labeled methemoglobin

The behaviour of the parameter A z z and of the trace of the hyperfine tensor,

1 / 3 T r A , with temperature for the w e t and lyophilized samples is shown in Fig. 4.

The value Azz of all samples decreases with increasing temperature. A b o v e 190 K

the rate of change of A z z of the wet samples is greater than that of the lyophilized

sample and increases with increasing temperature. However, the trace of the

hyperfine tensors of all samples is independent of temperature in the whole

temperature range investigated even in the critical temperature range between 220

and 300 K. However, the hyperfine separation as well as 1 / 3 T r A of the lyophilized

sample are less than the corresponding values of the wet samples. For a better

understanding of this behaviour the hyperfine tensor __Aof the spin label J A A 6 was

determined in model experiments in environments of different polarity at 120 K.

Fig. 5 shows the plot of 1 / 3 T r A against A z z for these samples. There is good

245

Azz/MHz

o

106

10/,

/',

102

,LA

100

98

96

80 ~oo ,20 ,,o 40 ,8o ~oo 220 2,0 2~o 28o ~/K

lira

_ _ _ "b , . : i

MHz

5O

~2

I I

80 100 120 lt,0 160 180 200 220 240 260 280 T/K

Fig. 4. (a) The parameter Azz as a function of temperature for methemoglobin-JAA6, which has been

immobilized by different methods: (A) frozen in aqueous solution, (z~) crystallized in a m m o n i u m sulfate

solution of high ionic strength, (O) lyophilized. (b) The trace of the hyperfine tensor, 1 / 3 TrA, as a

function of temperature for the same samples shown in (a).

linear correlation between 1 / 3 TrA and Azz and the values of the spin label bound

to the methemoglobin molecule fit the curve well. So we conclude that the

correlated change of Azz and 1 / 3 TrA by removing the water from the methe-

moglobin-JAA6 is due to a change of the polarity of the spin-label environment. On

the other hand, the temperature dependent change of Azz of all samples is not

correlated with a change of the trace of the tensor, the trace is even independent of

temperature in the whole temperature range investigated. Therefore a temperature-

induced change of the polarity of the spin-label environment in the protein can be

246

~-Tr A

MHz

50

48

z,8

44

42

~0 I I l l I t

Fig. 5. Correlation between the trace of the hyperfme tensor, 1/3 TrA, and Azz for the spin label JAA6

in systems of different polarity, determined at 120 K: ten) butanol, (11) ethanol, (o) lyophilizcd

methemoglobin-JAA6, (e) glycerol, (zx) methemoglobin-JAA6 in aqueous solution, (zx) crystallized

methemoglobin-JAA6.

e x c l u d e d a n d t h e o n l y e x p l a n a t i o n f o r t h e a b o v e r e s u l t s is t h e e x i s t e n c e o f a n i s o -

t r o p i c m o t i o n a l f l u c t u a t i o n s o f t h e l a b e l s e v e n in t h e f r o z e n s a m p l e s . A d e t a i l e d

a n a l y s i s o f t h e s e f l u c t u a t i o n s will b e g i v e n in a s u c c e s s i v e p a p e r .

A method using ni',roxide radical spin labels for determining both the isotropic rotational correlation

time r R and the environmental polarity of the label is described. By means of a least square fitting

method an effective hyperfine tensor ,4' and an effective g value tensor g ' are determined from X-band

EPR spectra. The traces of these tensors deliver the information about the environmental polarity of the

label and are not dependent on the rotational correlation time rg in the time range investigated from

10 ~ to 10 - m s. An averaging parameter S(rR) calculated from the values of the effective tensors

permits the evaluation of the rotational correlation time from experimental EPR spectra in the whole

time range given above. Due to the algorithm used here these informations about spin labels and

spin-labeled biomolecules may be evaluated from their EPR spectra using a personal computer. This

method may be applied to investigations of spin-labeled biomotecules where a change of motional

freedom of the label due to a structural change may be accompanied by a change of environmental

polarity. In this case the only consideration of the hyperfine separation would lead to incorrect results.

Acknowledgements

S p e c i a l t h a n k s a r e d u e to P r o f e s s o r A. R e d h a r d t f o r t h e n u m e r o u s i n s p i r i n g

c o n v e r s a t i o n s . T h e a s s i s t a n c e o f C. B a s s a r i s a n d H. G r o t t h a u s m a n n i n t h e p r e p a r a -

t i o n o f t h e m a n u s c r i p t is g r a t e f u l l y a c k n o w l e d g e d .

247

References

1 Stone, T.J., Buckman. T., Nordio, P.L. and McConnell, H.M. (1965) Proc. Natl. Acad. Sci. USA 54,

1010-1017

2 Freed, J.H (1976) in: Berliner, L.J. (Ed.), Spin Labeling TheoD' and Applications, Academic Press,

New York, pp. 53-132.

3 tlyde. J.S., Smigel, M.D., Dalton, L.R. and Dalton. L.A. (1975) J. Chem. Phys. 62, 1655-1667

4 Johnson, M.E. (1978) Biochemist~' 17, 1223-1228

5 Johnson, M.E. (1979) Biochemist~' 18, 378-384

6 McConnell, H.M. and McFarland, B.G. (1970) Q. Rev. Biophys. 3, 91-136

7 Lassmann, G., Ebert, B., Kuznetsov, A.N. and Damerau. W. (1973) Biochim. Biophys. Acta. 310,

298-304

8 Johnson. M.E. (1981) Biochemist~" 20, 3319-3328

9 Hwang, J.S.. Mason, R.P., Hwang. L.P. and Freed, J.H. (1975) J. Phys. Chem. 79, 489-511

10 Ruggiero. J.. Sanches, R., Tabak, M. and Nascimento, O.R. (1986) Can. J. Chem. 64. 366-372

11 Van, S.P., Birrell, G.B. and Griffith. O.H. (1974) J. Magn. Res. 15. 444-459

12 Bcnesch, R.E., Benesch, R., Renthal, R.D. and Maeda, N. (1972) Biochemist~, 11, 3576-3582

!3 McConnell, H.M., Deal, W. and Ogata, R.T. (1969) BiochemistD' 8, 2580-2585

24 PerutT, M.F. (1968) J. Cryst. Growth 2, 54-56

]5 Libertini. L.J. and Griffith, O.H. (1970) J. Chem. Phys. 53, ]359-1367

16 Weft, H.A. and Hecht. H.G. (1963) J. Chem. Phys. 38. 281-282

17 Braess. D. (1966) Computing 1,264-267

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