NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease

NMR analysis of the residual structure

in the denatured mutant of staphylococcal nuclease

state of an unusual David Shortle* Department

of Biological

and Chitrananda

Chemistry,

The

Johns

Hopkins

Background: Staphylococcal nuclease is a well-developed model system for analyzing the effects of mutations on protein folding and stability. Substitution of glycine 88 with valine (Gly88Val) destabilizes staphylococcal nuclease by 1 .Okcal mole- 1 and reduces its sensitivity to the denaturant guanidine hydrochloride, a phenomenon which may indicate an increase in residual structure in the denatured state. To assess its effects on denatured state structure, the Gly88Val mutation was incorporated into a 136 residue nonsense fragment which has been developed as a model of the wild type denatured state. Results: Application of two- and three-dimensional NMR spectroscopy to the Gly88Val fragment uniformly labeled with t5N and 3% has led to the assignment of 93 of the 136 residues. Comparison of chemical shifts of Structure Key

words:

folding

15

intermediate,

School

of Medicine,

Baltimore,

MD

21205,

USA

backbone resonances to those of wild type native nuclease, analysis of the secondary shifts of the assigned resonances and nuclear Overhauser effects involving backbone protons indicate that, unlike the wild type fragment, most if not all of the five-stranded P-barrel structure persists in this denatured state. Conclusion: One major effect of the Gly88Val mutation is to perturb the cooperative breakdown of the folded conformation, leading to a denatured state which is both more ordered and more stable than that formed by the wild type sequence. Since the equilibrium between the native and denatured states depends on the free energy difference between them, stabilization of the denatured state by the Gly88Val mutation indirectly destabilizes the native state.

October

Introduction The combination of site-directed mutagenesis and protein over-expression has made it possible to analyze the contributions of individual amino acid residues to the functional and structural properties of a large number of proteins. After a mutant protein has been constructed and purified and the differences between its behavior and that of wild type (wT) have been quantitated, explanations for the observed differences (or the lack thereof) are proposed that take into account the structural and chemical properties of WT and mutant side chains. This molecular dissection approach has been extensively used in attempts to understand the physical and chemical determinants of protein structures and their stabilities [ 1,2]. In virtually all cases, the emphasis is on the effect of the mutation on interactions that occur in the native or folded state [3], since the structure of this state is usually known in great detail. Yet such analyses overlook half of the equilibrium folding equation. The stability of the native structure is determined by the free energy difference between it and the non-native structure to which it breaks down, This non-native structure, which is usually referred to as the denatured state, is poorly understood because the available techniques for characterizing it do not reveal structure at the molecular level. Nevertheless, in principle, changing the free energy of the denatured state can change the equilibrium between it and the native state. An analysis of stability mutations in staphylococcal nuclease, a small 149 residue protein which *Corresponding

University

Abeygunawardana

1993, protein

1:121-134 folding,

protein

stability

has been extensively developed as a model system to study protein folding, has led us to conclude that muta tional effects on the structure of the denatured state are quite common [4,5], being found in more than half of all mutations studied [ 5,6]. Furthermore, these effects on denatured state structure appear to be energetically important, accounting for approximately half of the stability loss which accompanies an average mutation in staphylococcal nuclease [ 5,6]. To analyze the effects of mutations on the denatured state structure of staphylococcal nuclease under a wide variety of conditions, a very large nonsense fragment spanning residues 1 to 136 (1 +I36) was developed as a model denatured state [ 71. In the presence of substrate or tight-binding ligands, this fragment, which lacks five structural amino acid residues plus eight residues in an unstructured floppy tail, folds into a structure which is indistinguishable by catalytic activity and by far ultraviolet circular dichroism (CD) spectroscopy from WT nuclease. In the absence of such ligands, it unfolds to a relatively compact denatured state at physiological temperatures and pH [7]. We have used this nonsense fragment to study the effect of valine substitution for the WT glycine at position 88. This mutant was one of the first identified as altering the structure (and presumably the free energy) of the denatured state [4]. Initial characterization of its effects on the 1 -+ 136 fragment model of the denatured state clearly indicated that it increased the persistent, resid ual structure. Upon addition of denaturants such as

author.

@

Current

Biology

Ltd

ISSN

0969-2126

121

122

Structure

1993,

Vol

1 No

2

guanidine hydrochloride (GuHc~) or urea, this residual structure breaks down in a very broad and apparently cooperative transition as monitored by CD in the far ultraviolet and by gel filtration chromatography [7]. In this report, we describe a detailed investigation based on NMR spectroscopy into the residual structtre stabilized by the Gly88Val substitution,

Results Residual

structure

in Gly88Val(1-+136)

To assess the amount of residual structure that persists in the nuclease 1+ 136 fragment for both the WT sequence and the Gly88Val mutation, the CD spectra in the far ultraviolet region were determined [ 71. As can be seen in Fig. la, both the WT and the Gly88Val forms

of this fragment exhibit spectra significantly different from that expected of a random coil, with the Gly88Val mutation leading to an increase in elliptic&y of approximately 25 % in the region from 2lS23O nm. The one-dimensional proton NMR spectra of these two fragments show clear differences (Fig. lb). Whereas the WT fragment shows relatively little dispersion, the Gly88Val fragment spectrum reveals a number of upfield methyl groups, H, protons shifted downfield from the water resonance, and H, protons with chemical shifts downfield of 8.8 ppm, all of which are considered indicators of fixed, native-like protein structure. The l5N-fH correlation spectra for the two fragments more clearly reveal the large difference in the dispersion of the amide protons (Fig. 2). Whereas nearly all of the backbone amide protons of WT (1 -+ 136) are

(a)

J

190

I

I

I

I

I

200

210

220

230

240

250

Nanometers

6)

Fie. 1. (a) Far ultraviolet circular dichroism of Cly88Val (I -1361, spectra WT(1+136) and trypsin-digested staphylococcal nuclease. Data taken with permission from reference [71. (b) One-dimensional proton NMR spectrum of WT(1-+136) (lower trace) and GIy%8Val(1+136) (upper trace).

NMR

analysis

confined to the ‘random coil’ region between 8.1 and 8.7 ppm (Fig. Za), those of Gly8SVal(1-+136) are dispersed from 6.8 to lO.lOppm (Fig. 2b), a range that is consistent with a globular protein containing both a-helices and P-sheets. As has been noted by others [8,9] the chemical shift dispersion of the attached 15N nuclei remains high in denatured proteins, nearly comparable to that seen in the native state. The reasons for this are not understood. However, the 15N-lH correlation spectrum of Gly8SVal(l-136) shows two features that are very atypical for small globular‘proteins; a very large range in the intensities of individual peaks, and a disproportionately large number of peaks in the 8.1-8.7ppm range (Fig. 3a). As shown in an enlargement of the center of this spectrum (Fig. 3b), backbone amide resonances vary in intensity by at least a factor of 50, indicating large differences in the 15N and/or H, transverse relax ation rates (1/T2) of amides in different regions along the polypeptide chain and thus, presumably, in their motional behavior. All of the sharpest peaks except one (which is shown to be the carboxyl terminus) fall in the center of the random coil range, between 8.2 and 8.5ppm. In addition, many of the weakest peaks also fall in this same region of the spectrum, leading to severe crowding and considerable spectral overlap. Assignment of backbone Specific labeling with %‘J

resonances and 1% amino

of a denatured

protein

Shortle

and

Abeygunawardana

[lO,ll] were chosen to assign the backbone resonances and connect the amino acid spin systems. As described below, many of the amide proton resonances in Gly8SVal(l+ 136) are very weak, rendering the l5Ntotal correlation spectroscopy (TOCSY)-heteronuclear multiquantum correlation (HMQC) experiment virtu ally useless for defining the spin system type. Consequently, extensive use was made of specifically l5N- and r’&labeled amino acids to define the amino acid type of individual peaks in the IsN-lH correlation spectrum.

The heteronuclear single quantum correlation (HSQC) spectrum of Gly88Val(l-136) was obtained after labeling with each of the following amino acids: l5N-l~. sine, 15N-tyrosine, 15N-glycine, 1sN-leucine, 15N-&ine, 1sN-alanine, 15N-phenylalanine, l%glutamic acid and 1%aspartic acid. In all cases except that of lysine, cross-labeling of other amino acids occurred. For most amino acid types, this cross-labeling was relatively low and did not cause difficulty in correctly identifying the amino acid type of the most intensely labeled peaks. However, the peaks labeled by glutamic acid and aspartic acid were indistinguishable [12]. Because the 15N-lH correlation spectrum contains a number of very intense peaks and a number of incompletely resolved weak peaks, some of the residue type assignments were considered tentative because of the background of cross-labeling. The occurrence of faint crosslabeled peaks made it impossible to count precisely the number of correctly labeled peaks, but in every case except glutamic and aspartic acids, the number of strongly labeled peaks (relative to their intensity in a uniformly labeled sample) agreed to within f 1 or

acids

Because of the very poor dispersion for a large fraction of the amide protons and the unfortunate mix ture of very weak and very sharp peaks with similar proton chemical shifts, the methods using heteronu clear scalar couplings developed by Bax and colleagues

0

WT(l

(a)

+

136)

L 0 a IO’. 0

9.‘5

9:o

8.5 lH

Fig. 2. JsN-‘H

correlation

8.0

7:5

7.0

10.0

9.5

0 9.0

8.5 +I

(w-4

spectrum

(HSQC

method)

of (a) Wr(l

+I

36) and

E

0

(b) GIy88Val

(I -136)

8.’ 0 (w-4

fragments

7.#5

7.0

123

124

Structure

1993,

Vol

1 No

2

a)

M26 1 A69

d

K?O

,5

/l K26

K24

I 9.4

I 9.2

8-I72

Ail2

Lg

D

417

L I 9.6

I I 2 10.0 9.8

.a-w-7 I 9.0

I 8.8

I 8.6

I 8.4

I 8.2

I 8.0

I 7.8

I 7.6

I 7.4

I 7.2

I, 7.0

6.8

‘H (ppm) (b)

K

D/E Nll8

-

Nl19 I hlCcl 3’

U-L,

H112l- R87

Q80

-VIII

083 1

k63

Fig. 3. (a) 15N-lH

correlation spectrum (HSQC method) of Gly88Val (I -1361, with sequence-specific assignments labeled using the one-letter amino acid code. Peaks identified by specific amino acid labeling but not assigned to a unique position in the amino acid sequence are labeled by amino acid type. (b) Enlargement of the central portion of the l5N-lH correlation spectrum.

K136 ,

,

,

,

I

I

8.6

8.4

8.2

8.0

7.8

7. 6

‘H (ppm)

2 with the number predicted from the amino acid sequence. To identify peaks corresponding to methionine, threonine, arginine and histidine, for which 1%labeled preparations are either unavailable or very expensive, each of these ammo acids in the natural (l*N-labeled) form was added singly to bacterial cultures grown on 15N-ammonium chloride. By identifying the missing peaks in the the 15NP1H correlation spectra, 7 of the 9 threonines, 3 of the 5 arginines, and 2 of the 4 histidines were identified. In the case of methionine, the

‘bleaching’ of the correlation peaks was not complete; 2 of the 4 methionines were tentatively assigned on the basis of - 75 % reduction in intensity. Those spin systems whose amino acid types were established by specific labeling are shown in Fig. 4. Double

and

triple

resonance

experiments

A 15N-TOCSYmHMQC experiment was used to deter mine the chemical shifts of the H, and side chain protons. For more than 3/4 of the spin systems, the only cross peak observed was from the H, proton. Thus, as

NMR

IO 15N/14N

Labeling

N/

14

---II-m -I m I

H,(i) -C&l) H,(i) -CO(i-1) H,(i) -N(i+l) H&-H,(i-1) H,(i)-H&l)

II

=

III m-m

I II

I

I -

70

80

90 YG

VLAY

III -

II

II

I I

100 Labeling

40

GVBKYG AF KK V AKKLEV F KGBRT PKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGORTDKYGRVLAYI

HN(i) -C,(i-1) H,(i) -CO(i -1) H,(i) -N(i+l) H&)-H&l) H,(i)+HN(i-1)

“Nt4N

30

60

50 N Labeling

of a denatured

ALBGBTVKL YKGB ATSTKKL~~EP~::IKAIDGDTVKLMYKGOPM~~~~LLVOTPETK~

H,(i) -C,(i-1) H,(i) -CO(i -1) H,(i) -N(i+l) H,(i)-H,(i-1) H,(i)-H,(i-1)

15

20

analysis

I

111 m--III

I

130

120

110

--m-m mmI-I I

II -

I.

VAYVYK BBTHBB YADk%VNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAOAKKEK I I I

I

WI

LRKG

ABAKKBK

I-II -I

I -I

m

is frequently the case for relatively large globular proteins, this experiment was of little help in defining the spin system and thus the amino acid type. To obtain the chemical shift of the preceding H, and to detect structurally informative nuclear Overhauser effects (NOES) to the amide protons, an 15N-nuclear Overhauser effect spectroscopy (NOESY)HMQC was carried out. Connectivities between H,(i - 1) and HN(i) for 49 spin systems were identified; in addition, a small number of HN-HN NOES, which often characterize a-helices and sharp turns, were found. These NOES are shown in Fig. 4, which summarizes all of the data used in establishing the assignment of backbone resonances. The most useful triple resonance experiments for establishing connections between adjacent spin sys terns were the amide proton to nitrogen to cl-carbon (HNCA) and amide proton to nitrogen to a-carbon (via carbonyl) [ HN(CO)CA] correlation pair. For a spin system defined by a peak in the 15N-lH correlation spectrum, the former experiment yields the chemical shift of the t3C, nuclei of the same spin system and in a majority of cases that of the preceding spin system as well. The latter experiment yields the chemical shift of the W, nucleus of the preceding spin system only, allowing missing or uncertain inter-residue connectivities from the HNCA experiment to be determined. Fig. 5 shows the sequential connections defined by these two experiments as a trace through the HNCA spectrum extending from Lys70 to Asp83. Of the 93 assigned residues, sequential connectivities involving the a-carbon could be traced for 77. Additional connections between adjacent residues were also established by linking the 13C-carbonyl nucleus via the amide proton to nitrogen to carbony1 (HNCO) and a-proton to cc-carbon to carbonyl (HCACO) correlation pair of experiments and on the basis of the t5N nucleus via the a-proton to a-carbon to nitrogen (via carbonyl) [HCA(CO)N] correla

II-I I

protein

Shortle

and

Abeygunawardana

Fig. 4. A schematic summary of data used in assigning the backbone spin systems in Cly88Val (I -136). Identified above the amino acid sequence are residue type assignments (in the one letter code) established by specific lsN or 14N amino acid labeling and analysis of the {‘H,15 N} HSQC spectra. The letter ‘B’ indicates labeling by both 15N-glutamic acid and 14N-aspartic acid. A black bar connecting two adjacent residues indicates that the correlation listed on the left was observed. The H,(i)+C,(i - 1) connectivity was established by the HNCA and HN(CO)CA experiments; the HN(i)+CO(i1) connectivity by the HNCO and HCACO experiments (which is based on the H, and C, assignments obtained from the TOCSY-HMQC and the HNCA experiments); the H,(i)+N(i + I) connectivity by the HCA(CO)N experiment; and the HN(i)+HW(i-l) and the HN(i)-+HN(i-I) by the 15N-NOESY-HMQC experiment.

tion experiment, In both cases, fewer than 50 % of spin systems could be connected because of overlap in the. chemical shifts of the H, proton and of 13C, for many of the spin systems upon comparing data sets collected in Hz0 and D,O. This problem is also encountered with globular proteins [13] and makes it difficult definitively to establish the identity of cross peaks corresponding to the WO of the same residue and the 15N of the following residue. Nevertheless, for 56 and 29 residues respectively, these two connectitities were identified, increasing the confidence level of the assignments that could be made. With this set of experiments, a total of 93 out of 136 backbone spin systems could be assigned with co& dence; values of chemical shifts are listed in Table 1, As can be seen in Fig. 4, there are three major stretches of polypeptide chain that could not be assigned: 3649, 5659, and ‘$3110. The first of these corresponds to the end of the third P-strand in native nuclease, followed by a very hydrophobic stretch of residues and a flexible loop that forms part of the active site. Residues 42-49 have proven very difficult to assign for the WI native protein [12,14]. Residues 5659 form the first turn of the first a-helix and residues 9GllO form the second cl-helix followed by a tight turn and the beginning of an extended chain segment. Structural

analysis

The determination of protein structure at high resolution requires assignment of side chain protons and identification of NOES between side chains and between side chains and H, and H, protons. This type of analysis can only proceed with confidence after essentially all spin systems have been identified, a task which may not be achievable for GlySWal(1+136) because of the extremely short Tzs of many of the H,, H,, N and C, resonances and because of severe overlap in the 15N-lH and t3C-lH correlation spectra. Nevertheless, secondary structure and low resolution tertiary inter-

125

126

Structure

1993,

K70

K71

Fig. 5. A trace each

lane,

Vol

the

of the larger

1 No

172

2

E73

connections marked peak

V74

established represents

E75

F76

D77 K78 G79 Q80 R81 l-82

by the HNCA experiment the C,(i) resonance.

actions can often be identified from NOES involving backbone protons. In addition, it has recently become clear that the chemical shifts of both the H, proton and the t3C, carbon are very strongly correlated with secondary structure [ 15-l 71. We found that many of the chemical shifts of assigned backbone resonances correspond quite closely with those of native WT nuclease. For example, Lys70 in native WT nuclease has been assigned chemical shifts for H,, N, H, and C, of {10.14,128.0,4.38,57.1} by Wang et al. [14] (after addition of the difference in the average 15N chemical shift ( + 1.7 ppm) and 1% chemical shift (+OBppm) over all assigned residues to correct for referencing offsets). In Gly88Val(l-+ 136), this residue has corresponding val ues of {10.11,127.6,4.35,57.7}. Another highly similar residue is Met26: {9.54,124.8,4.89,54.2} in folded, na tive nuclease and {9.58,124.2,4.80,54.5} in the denatured fragment. To quantitate the similarity in chemical shift values between the assigned residues in Gly88Val(l+ 136) and those in WT nuclease, the general similarity measure of Gower [18] was calculated. In this case the similarity measure s is defined as:

between

HN(i)

and

s = 1 ~ (IHN+

C,(i - I) extending

- HN,,~

I/l.00

+ l”N,

083

from

Lys70

-15

to Asp83.

NJ/15.0

In

+

b,, - H,,,,l/l.OO+ 113Ca,p -13C,,,,l/6.0)/4

where u designates the chemical shift for Gly88Val(l-+ 136) and wt designates wild type. For H,, l5N and 1% values, the denominator is the maximum observed difference in chemical shifts between the two proteins for any residue, and for H,, where the maximum difference is 2.71, 1.00 is used to prevent underweighting of this value. Thus, s is approximately equally weighted for each of the four nuclei, with a value of 1.0 indicating identity and a value of 0.0 indicating the most extreme dissimilarity possible, For both Lys70 and Met26, s is equal to 0.95. The value of s was determined for all residues for which at least three of these four chemical shifts had been determined, both for Gly88Val(1-+136) and for native nuclease [ 141. In Fig. 6, those residues where s falls between 0.80 and 0.90 are marked with an open circle, whereas residues with a value of s greater than 0.90 are marked with a filled circle. Fig. 6 also shows a linear arrangement of the elements of secondary structure found in native nu-

NMR

'able

1. Chemical

Residue

shifts of backbone

'HN

15N

resonances

in C88V

analysis

of a denatured

protein

Shortle

and

Abeygunawardana

(1+136).

%Z

736

13co

Residue

'HN

l5N

'HE

'3C,

'3CO

Leu7 His8

8.18 8.57

121.0

4.17 4.80

54.4 54.82

176.27 173.35

Gill75 Phe76

8.95 8.57

127.1 124.2

4.96 4.98

54.85 56.36

174.28 173.31

Lys9 clulo Pro11

8.31 9.03

125.2 125.3

4.30 5.01 4.95

56.25 53.5 63.2

176.2 173.21 175.28

Asp77 Lys78 Cly79

8.93 8.72 8.63

123.0 128.1 109.9

4.66 4.19 3.75

53.23 57.9 45.14

177.13 177.17 -

Ala12

8.11

122.1

4.97

51.46

175.22

Cln80

7.67

121.4

4.23

55.62

Thr13 Leul4 lle15

8.11 9.19 8.01

111.6 127.6 116.9

4.65 4.07 4.27

60.47 56.84 62.09

173.9 175.95 174.14

Arg81 Thr82 Asp83

8.49 8.10 8.39

124.7 114.8 122.4

4.44 4.67 4.64

56.18 60.71 52.73

176.78 175.08 174.84

Lys16 Ala17

9.57

132.5

4.36 4.50

56.47 52.05

173.78 175.26

Tyr85 Cly86

7.95 8.12

120.5 110.7

4.59 4.18

56.68 45.45

175.9 -

lIeI Asp19

8.02 8.23

127.1 120.4

3.94 4.49

63.17 53.71

175.33 176.4

Arg87 Va188

8.42 8.03

122.4 123.9

4.35 3.96

55.58 61.53

175.47

Cly20 Asp21 Thr22

8.54 7.99 7.58

105.6 114.6 117.8

4.88 5.40

47.2 52.13 62.18

175.43 173.9

Leu89 Ala90 Tyr91

8.54 8.67 8.84

131.5 124.8 121.9

4.90 4.87 5.64

54.97 51.38 57.77

177.75 176.4 -

Val23 Lys24 Leu25

9.00 9.32 9.36

123.3 129.4 128.7

4.57 5.83 5.16

60.22 55.44 52.91

172.09 175.33 174.21

Ala94 Asp95 Cly96

9.76 9.39

129.1 105.3

4.98 4.39 4.20

49.59 56.38 45.17

175.65 176.01 -

Met26

9.58

124.2

4.80

54.46

174.93

Lys97

7.84

123.2

4.64

54.31

175.86

Tyr27 Lys28 Cly29

9.13 9.33 8.41

132.5 129.4 104.3

4.92 3.57 4.10

56.88 57.3 45.48

173.61 176.77 -

Vallll Ala112 Tjirll3

7.97 8.27 8.07

122.1 129.4 122.1

4.07 4.26 4.49

61.99 52.04 57.8

175.25 176.74 174.97

Cln30 Thr33 Phe34

7.93

121.8

9.09

127.8

4.99 4.55 5.15

52.08 63.15 56.34

173.2 172.4 173.91

Val114 Tyrll5 Lysll6

7.87 a.27 8.03

125.2 127.5 128.3

3.99 4.45 4.47

61.69 57.81 53.34

174.72 174.69 173.42

Arg35 Cly50 Val51

9.00 8.47 7.99

124.6 113.0 120.9

5.42 3.95 4.07

52.33 45.15 62.14

175.18 .175.82

Pro117 Asnll8 AsnIl

~ 8.49 a.37

120.8 121.6

4.28 4.57 4.75

63.08 53.05 53.05

176.38 174.84 175.43

Clu52 Lys53 Tyr54 Cly55

8.51 8.15 8.24 8.41

126.6 123.7 122.2 111.3

4.21 4.17 4.63 4.20

56.41 56.18 57.74 45.27

175.92 176.13 -.

Thrl20 His121 Clu122 Cln123

8.11 8.48 8.31 8.39

116.1 122.3 123.7 123.4

4.21 4.66 4.19 4.22

62.3 55.64 56.64 55.93

174.54 174.55 176.26 175.7

Ala60 Phebl

8.18 8.03

126.5 120.4

4.21 4.58

52.82 58.05

176.13

His124 Leu125

8.25

125.3

4.11 4.27

55.03 55.1

179.31 177.1

Thr62 Lys63 Lys64

7.88 7.69 7.61

117.5 121.9 119.1

4.16 3.53 3.91

62.41 60.07 59.01

176.04 176.88 178.56

Arg126 Lys127 Ser128

8.38 8.28 8.30

124.4 126.0 125.2

4.31 4.30 4.30

55.98 56.27 56.0

176.2 -

Met65

7.72

118.6

3.87

59.18

179.01

cm29

8.42

124.9

4.26

56.56

175.23

Val66 Asn68 Ala69

8.15 8.30 6.78

125.5 120.7 123.9

4.17 4.94 4.45

53.16 51.79

172.28 177.75

Ala130 Cln131 Ala132

a.25 8.21 8.22

126.5 121.2 127.2

4.24 4.25 4.25

52.79 55.74 52.51

177.82 176.16 177.48

Lys70 Lys71 lle71 Clu73

10.11 8.92 8.99 8.79

127.6 123.7 130.7 125.5

4.35 4.63 5.22 5.28

57.7 55.33 58.55 53.66

177.7 175.08 175.4 174.56

Lys133 Lys134 Clu135 Lys136

a.20 8.39 8.45 7.88

122.8 125.4 125.2 128.4

4.25 4.27 4.27 4.07

56.17 56.52 56.62 57.72

176.54 175.23 181.05

Val74

9.63

119.0

5.23

58.94

173.81

clease, plus two indicators of secondary structure in GlyS8Val (l-136): the H, chemical shift index [ 161 and the t3C, secondary shifts [15,17], defined by the difference between the observed chemical shift and that of the same residue in a random coil. The residue stretches showing high similarity to native nuclease include the chain segments that form P-strands 1, 2 and 4, the carboxyl terminus of helix 1 and tight turns involving residues 85-87 and 95-97, suggesting that these portions of the denatured state may have nativelike structure. In addition, the correspondence between chain segments that form P-sheet in native nuclease and those that show several consecutive H, protons with ‘P-type’ chemical shift indices is nearly perfect.

P-strands 2 and 4 ali gn exactly with the chemical shift index pattern, and strands 3 and 5, both of which have two unassigned residues near the middle, also show good agreement, In native nuclease, b-strand 1 contains a P-bulge residue at position I6 and shows a similar broken pattern of P-like chemical shift indices. Similary, the 1X, secondary shifts in Gly88Val (1 + 136) support the same patterns of P-sheet structure, namely those chain segments which correspond to P-strands in native nuclease. Also shown in Fig. 6 are the positions of the three ahelices. Assignments are missing for the amino-terminal half of the first a-helix, but the H, chemical shift indices

127

128

Structure

1993,

Vol

1 No

M

native

2” structure

M

native

2” structure

2

110

100

120

130

YADGI
H,

+-

______

-_---

CU

WT

native

2” structure

Fig. 6. A chart

of the H, chemical shift index and the secondary shifts of the C, nuclei with respect to the secondary structure elements in WT native nuclease. An open circle beneath the residue indicates a similarity index s (defined in text) between 0.80 and 0.90 for the chemical shifts of H,, H,, N, and C, of that residue compared to WT native nuclease; a filled circle indicates s is greater than 0.90. For the H, chemical shift index, a downward pointing line signifies the H, resonance of the residue was shifted downfield from its random coil value by more than 0.1 ppm, indicating a possible p-sheet type conformation 1161. An upward pointing line signifies the H, resonance is shifted upfield by more than 0.1 ppm, indicating a possible a-helical conformation, and a solid dot indicates a shift < f 0.1 ppm. For the C, secondary shifts calculated using the random coil values in reference 1171, a bar below the line signifies an upfield shift, indicating a possible p-sheet conformation, whereas a bar above the line signifies a downfield shift, indicating a possible cc-helical conformation. The five P-strands (arrows), three a-helices, and several tight turns observed in folded WT nuclease are indicated below showing their relative positions with respect to the amino acid sequence.

and the 13C, secondary shifts both indicate that the carboxy-terminal half is helical in Gly88Val(l --f 136). Unfortunately, the major missing block of assigned residues includes the second a-helix, so no information is available on its local structure. But for the third a-helix, both chemical shift parameters suggest that no significant amount of helical structure persists. In fact, this segment of the protein appears to be without strutture. Residues 123-136 all exhibit very sharp peaks in the 15N-lH correlation spectrum, suggesting that this part of the polypeptide chain is highly mobile. (Several analyses of samples by SDS gel electrophoresis after data collection clearly indicated that these sharp lines were not a result of partial proteolysis.) Perhaps the most characteristic NMR feature of antiparallel P-sheets is the presence of relatively strong NOES between H, protons. At least five NOE cross peaks are evident between protons with chemical shifts between 4.5 and 5.5ppm (Fig. 7). Since a number of residues have not been assigned, including Pro31, Met32, Ile92 and Tyr93 which may be in P-sheets (and

therefore have H, chemical shifts in this range), these NOES cannot be unambiguously ascribed to unique residues. However, the NOE at 5.45-4.53ppm is consistent with a Lys24-Thr33 connectivity, the NOE at 5.29-4.96/4.99 ppm is consistent with a ProllGlu73 connectivity, and the NOE at 5.00-4.90ppm is consistent with a Phe7GAla90 connectivity. Since these connectivities correspond to 3 of the 10 seen in native nuclease [ 121, it seems highly probable that these tenative assignments are correct. If so, these three NOES would define native-like hydrogen bonding between strands 2-3, l-4 and 4-5. Further evidence for native-like structure includes patterns of strong local and medium range HN-HN NOES (Fig. 8). In WT folded nuclease, residues 19-22 form a type I turn, residues 27-30 a I’ turn, residues 84-87 a type I turn and residues 94-97 a type I’ turn; all four turns display the expected backbone NOES in native nuclease [14]. The HrHN NOES shown in Fig. 8 are entirely consistent with the presence of native

NMR

P 5.6

I

I

5.4

/

5.2

I

I

5.0 (w-n

analysis

of a denatured

4.6

)

or nearly native turn structures in GlyS8Val (1 + 136) at these same four positions. While this structural analysis is not complete, the data presented above suggest that the basic organization of the five-stranded P-barrel, which encloses the major hydrophobic core of the protein, persists in Gly88Val (l-136). The last two turns of the first a-helix, which are amphipathic, form a helical structure and thus appear to contribute the hydrophobic side chains Phe61, Thr62, Met65 and Val66 to this major hydrophobic core, as in the WT native structure. None of the spin systems in the second a-helix, which extends from residue 98 to 105, have been assigned. However, the type I’ turn that immediately precedes this helix is present, suggesting that at least part of the second ahelix may also be present. If so, the side chains of Val99 and Leu103 would be positioned to participate in the major hydrophobic core, as in the native structure. [NMR analysis of a second, less structured denatured state of staphylococcal nuclease clearly indicates that this helix is the most stable of all secondary structures, with residues 98-105 being in a helical state approximately 30 % of the time (A Alexandrescu, C Abeygu nawardana & D Shortle, unpublished data)]. There is no evidence that the third and last a-helix, formed by residues 123-136, is present; instead, the long TZs displayed by all of these residues strongly suggest that they have virtually unrestricted motion.

Shortle

and

Abeygunawardana

Fie. 7. Central region of the lH,jH NOESY spectrum, showing five candidate HuHor NOES, three of which are consistent with native-like pairing and 4 between P-strands 1 (5.29/4.96-4.99), 2 and 3 (5.4514.53) and 4 and 5 (5.0014.90).

II

4.8

protein

Several polypeptide segments connecting helices and strands give H, H N NOES suggestive of native-like structure (Fig. 8). However, no data constrain the structural state of segments LeuSGPheGI, Asp77-Tyr85 and Vallll-Lysl18. In each segment, those residues that have been assigned do not show obvious similarities in chemical shifts to the equivalent residues in the WT native structure nor do the NOE data or the chemical shift parameters indicate regular secondary structure. However, the line widths of the amide protons (Fig. 3) do suggest that these chain segments are not highly flexible. Since parts of each of these three chain segments interact in the native structure with residues 123-136, a segment which behaves as a flexible tail in Gly88Val(l-+ 136), the observed non-native chemical shifts presumably are a consequence of altered tertiary interactions, A ribbon diagram highlighting those secondary structures in Gly88Val (1 + 136) that appear to be native-like is shown in Fig. 9.

Discussion

The application of NMR spectroscopy to the structural analysis of partially folded proteins holds considerable promise for gaining insights into the energetics of protein folding [8,9,19,20]. The low resolution NMR characterization of the residual structure in a denatured protein reported here is one of the first of its kind car

129

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Structure

1993,

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1 No

2

. .

l

Gin 30 .

K.

.

k

Gly29 . . . af

Lys28

~II

. l

Asp19

. . l

Tvr27

.

l

l

. . . Tyr85

. . .
Arg87 .

ried out under non-denaturing conditions. One of the most surprising observations resulting from this study is the apparent disparity between the small structural differences between the WI and Gly8SVal fragments detected by CD and the very large change in amide chemical shift dispersion (Figs 1 and 2). The elliptic&y in the region between 210 and 230 nm, which includes the most prominent minima for both a-helices and pstrands, increases by approximately 25 %, suggesting that Gly88Val induces a relatively modest increase in secondary structure. But in the l5N-lH proton correlation spectra, it is observed that this substitution causes more than 25 peaks to move downfield or upfield from their random coil values (8.1~8.7ppm), and with the sequence-specific assignments, this set of shifted peaks includes residues assigned to each of the five P-strands and at least one of three a-helices present in the native structure.

. . %,,8{

Fig. 8. A diagram of four polypeptide segments in Cly88Val (I -136) where several strong H,-H, NOES were observed. The arrow indicates the direction of NOE transfer, with the arrow pointing to the proton detected during ta of the NOESY-HMQC spectrum. These regions correspond precisely with the positions of tight turns in folded WT nuclease and represent a subset of H,-H, NOES observed in the native solution structure I141.

The simplest way of resolving this paradox is to propose that the structure of the polypeptide chain in WT (1 -+ 136) is configured in space in more or less the same way as Gly8SVal(l+l36), but that its structure has a greater dynamic character. That is, the P-strands and a-helix 1 (and perhaps 2) of the WT fragment are present a significant fraction of the time and are arranged in the same topology as in the WI folded conformation, but these secondary structural elements undergo sufficient dynamic motion to average out the

Fig. 9. (a) Schematic diagram of the 3D structure of the native (WT) state of staphylococcal nuclease (drawn by Jane Richardson and used with her permission). The, position of residue 136, which corresponds to the carboxyl terminus of the (1+136) fragment, is marked by an arrow. (b) Schematic diagram showing the structural motifs present in Gly88Val (I +136) in the context of the structure of WT native nuclease. Red shading indicates that the corresponding chain segment has the same secondary structure in both the mutant denatured state and in the WT native state, and for the five p-strands, some of the sheet pairing interactions are present. Blue segments indicate structure present in WT native nuclease that has not been demonstrated in Gly88Val(1+136), either because of lack of structural information or because portions of the chain have not been assigned. The green shading indicates an unstructured, freely mobile region at the carboxyl terminus.

NMR

analysis

magnetic environment of the backbone protons, yielding random coil chemical shifts for the amide protons. Thus, the average $-+ angles for many residues may be approximately the same in the two fragments, yet the greater fluctuations away from these average values in WT (1 + 136) lead to a loss in chemical shift dispersion. It follows that amide proton chemical shifts are not reliable indicators of residual structure which has a dynamic and statistical character. In view of these clear qualitative differences, it is noteworthy that the residual structure of these two fragments exhibit one striking similarity: the cooperativity with which this persistent structure breaks down with increasing GuHCl concentration, as monitored by CD and gel filtration chromatography, is virtually identical for the two fragments [7]. In other words, the rates of decrease in the magnitude of elliptic&y at 222 nm and increase in hydrodynamic radius as functions of GuHCl concentration are the same for WT (l-136) and Gly88Val(l+ 136) fragments. When these observe tions were first made, they were explained by proposing first, the existence of two distinct denatured states ~ D,, which is highly structured, and D, with much less structure, and second, in the absence of GuHCl, the equilibrium for the WT fragment corresponds to -75 % D, and 25 % D2, whereas stabilization of D, by the Gly88Val mutation increases its concentration to > 95 %. In view of the sharp lines and lack of dispersion observed for WT (1 + 136), this explanation now seems less likely. One intriguing alternative is that the physical interactions underlying the structural cooperativity displayed by these fragments do not involve the tight packing and hydrogen bonding of rigid, native-like structure. Instead, this cooperativity may depend on one or more dynamic interactions that both fragments have in common, such as bulying hydrophobic surface. A second paradox is the finding that Gly88Va1, a mutation that destabilizes the native state, clearly stabilizes a structure in the denatured state that is very similar to the five-stranded P-barrel topology found in the native state. If this mutation stabilizes a portion of the native structure, why doesn’t it stabilize the native state? There is a simple though incomplete answer to this narrowly focused question: to the extent that proteins are perfectly cooperative ‘all or nothing’ systems, the structure stabilized in the denatured state of the Gly88Val mutant cannot be a precise subset of native structure. If it were, the native state would also be stabilized. Therefore, the structure svdbilized by Gly88Val(l+ 136) must be sufficiently different from native structure to interfere with the formation of the remaining subset of polypep tide chain structures, in particular those dependent on interactions with the five-stranded P-barrel. One possibility is that the valine side chain at position 88 cannot participate in the hydrophobic core in the native state, but upon denaturation the core is slightly distorted to allow packing of this hydrophobic group. A more complete explanation of the lower stability of nuclease in the presence of the Gly88Val mutation must

of a denatured

protein

Shortle and Abeygunawardana

deal with the free energy of its denatured state. From the data presented above it is Cklr that less of the structure breaks down when Gly88Val nuclease dellatures than when the WT enzyme does, In fact, much of the residual structure that persists in its denatured state is surprising!y native-like. In one sense, it can be said that nuclease Gly88Val undergoes a less coopera~ tive breakdown in structure ~ only part of the folded conformation is disrupted, with the undisrupted corn ponent constituting the major hydrophobic core of the molecule. Instead of the native state being ‘shattered into small pieces’ upon denaturation, for this mutant it only appears to be ‘cracked open’, perhaps yielding only two ‘pieces’. If the assumption is made that chain entropy and hydrophobic surface exposure represent the two terms which dominate the energetics of the denatured state [ 21,6], then the effects of the Gly88Val mutation can be explained as an alteration in the balance between these two opposing energy terms. The amount of hydrophobic surface exposed in the denatured state has been significantly reduced, lowering its free energy. However, the gain in chain entropy that accompanies denaturation has also been reduced, and this raises the free energy of the Gly88Val denatured state relative to that of WT. The difference in free energy between the Gly88Val and W’T denatured states will depend on the relative magnitudes of these two changes in free energy From an extensive study of stability mutants of staphylococcal nuclease and their effects on the sensitivity of nuclease to solvent denaturation, we have reached the conclusion that, for this protein, the amino acid sequence has evolved to confer on the denatured state some sort of maximal value of free energy 161. If this conclusion is correct, it follows that the Gly88Val de natured state is lower in free energy than the WT denatured state, and therefore that the loss in free energy that accompanies the reduced exposure of hydrophobic surface outweighs the gain in free energy that accompanies the reduction in the entropy of the denatured polypeptide chain. Similar arguments have been made to explain the paradoxical destabilization of the native state that occasionally accompanies the engi neering of a new disulfide crossbridge into a protein

[22,23],

Biological implications One major assumption on which much of modern protein science is based is that most, if not all, properties of proteins can be quantitatively understood by reference to their native, folded conformations. In this study, a single amino acid substitution that destabilizes the native state of staphylococcal nuclease has been shown to lead

131

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Structure

1993,

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to a profound increase in fixed, native-like structure in the denatured state. Considering the magnitude of these changes in denatured state structure, it seems likely that the stability loss caused by this mutation results primarily from a lowering of the free energy of the denatured state. The native state is thereby made less stable through the reduction in the free energy difference between the native and denatured states. From this line of reasoning, it follows that the structure of the folded, native conformation does not by itself contain sufficient information to explain its stability. A full quantitative understanding of protein stability will therefore require a much more complete characterization of the chain-chain interactions that occur in partially folded structural forms of proteins. As demonstrated in this study, NMR spectroscopy can be applied to the characterization of nonnative protein structure. Although it is not yet clear how high a resolution such structural analyses will eventually attain, they promise a wealth of new insights into the processes and energetits of self-organization of polypeptide chains - a new window on the physical principles underlying protein folding. These new insights will undoubtedly depend on new ways to define, characterize, and describe polypeptide structure that has a large statistical component.

Materials Protein

and methods preparation

The genes for the WT and Gly88Val mutant forms of staphylo~ coccal nuclease nonsense fragment (1 -136) were cloned into the pET12a expression vector and transformed into &chcrichiu coli strain HMS174 (DE3) (Novagen, Inc., Madison, WI). Uniform isotopic labeling with l3C and/or ]5N was carried out on a 350 ml culture of cells grown in MOPS media [ 241 supplemented with ‘~NH&I, with or without uniformly t%labeled glucose. For specific labeling with one ‘SN~amino acid, the culture was grown with l*NH,&l as the sole nitrogen source until nuclease expression was induced with .I mM isopropyl fl~Dthiogalacto side (IPTG), at which time the labeled amino acid was added to a final concentration of 50 mg I,- I. Specific labeling with one l*N~amino acid was done in a similar way, with the unlabeled amino acid added at the same time as the initiation of protein expression. Protein was purified by urea extraction, ethanol pre cipitation, and sulpho-Sepharose ion exchange chromatography as previously described [7], except that the halvested cells were pre-extracted with 6M urea, pH9.2 without added NdCI. This step solubilizes almost all cellular proteins without solubilizing the nuclease fragment present in inclusion bodies. After a final 12-18 h dialysis against deionized Il,O, the isolated protein WdS lyophilized and stored at - 70°C. NMR

containing 1 mM sodium azide and either 8 o/o D,O or 99.9 % D,O was adjusted in pH to a meter reading of 5.3. All NMR data were collected with a modiliecl Bruker AM600 spectrometer equipped with an external timer device and two additional het~ eronuclear RF channels driven by external synthesizers (Model 310, P’I’S Inc., Littleton, MA) phase locked to the 10 MHz spec trometer master clock as described in [lo]. The high speed RF gates/level switches, globally optimized alternating phase rectangular pulses (GARP)flALTZ modulators, and the AMTIMER used in these external channels were obtained from Tschudin Associates (Kensington, MD); the 25 and 50watt class A linear amplifiers were obtained from ENI, Inc. (Rochester, NH), and 13 dB booster amplifier from Calmus, Inc. NMR data sets were transferred to a Personal IRIS 4D/35 workstation and processed with the data analysis software FELIX (Hare Research, Inc. Woodinville, WA). 1H chemical shifts are reported with respect to the II,0 resonance, which is taken as 4.754ppm downfield from the external standard sodium 3~ (trimethylsilyl)propionate2,2,2,3,3-dd (TSP) in I),0 (defined as 0.00 ppm) at 27°C. “C chemical shifts are reported with respect to an external TSP standard in D,O (defined as O.Oppm). l5N chemical shifts are reported with respect to an external 15NH&l standard (2.9 mM in 1 M HCl) at 2O”C, which is considered to be 24.93 ppm downfield of NIJj [ 251,

20

NMR

spectroscopy

lH&tjN I ISQC spectra [26] were recorded in Hz0 using the pulse sequence of Messerle et al. [27] with a 2 ms purge pulse at the end of the first insensitive nuclei enhancement by polar~ ization transfer (INEPT) to suppress the water signal. IH Deb coupling was achieved with a 180” pulse in the middle of the t1 period, and ‘jN decoupling during acquisition (tZ) employed CARP-1 modulation of a 1.25KHz RF field [28]. Either 512 or 768 ti values were recorded using time proportional phase incrementation (TPPI) [ 291, with either 16 or 32 scans per tl value. The spectral width for ‘jN was 2193 Hz and the carrier frequency was set at 117.9ppm. Data were processed with a cosine bell filter in both dimensions and zero-filled to give data matrices of 1 K by 2 K real points. A ZD-NOESY spectrum [30] was recorded in 99.8 “/o D,O with a 100 ms mixing time. The spectral width was 8064 Hz in both dim mensions, with averaging of 32 scans per tl value. The spectrum was derived from a 1024 (real) x 4096 (real) data matrix, with acquisition times of 36.4 (t,) and 290 (tZ) ms. Atter applying cosine bell filter and zero filling before Fourier transformation, the final data tnatrix was 2048 (complex) x 4096 (complex). 31) NMK

spectroscopy

For all heteronuclear 3D experiments, quadrature data were colt lected in t, and tL by the States-TPPI method [31]. For spectra recorded in I-1,0, the solvent signal was suppressed with a weak presaturation pulse (field strength of 25Hz) during the relaxation delay. Residual solvent signal was removed from the final spectrum by convolution of the time domain data [32] during data processing, and for spectra based on detection of amide protons, the aliphatic half of the spectrum was discarded after zero-filling and Fourier transformation. Generally, a cosine bell filter was applied in tl and t2 after using linear prediction to extend the data by one third, while a sine bell filter with phase shift ranging from 40”-90” was applied to the t3 dimension. The data matrices were zero~filled by at least two prior to Fourier transformation to obtain the desired digital resolution.

spectroscopy

All NMR spectra were collected at 27°C on protein samples at concentrations between 1.3 mM and 4.0 mlM. The final solution,

The 3D {‘H,” N} TOCSY-IIMQC experiment pulse sequence and phase cycle of Pelton

[33] employed the et al. [34], with a

NMR

analysis

34 ms spin lock using decoupling in the presence of scalar interactions (DIP%2) sequence [35] plus a 17 ms delay to remove rotating frame NOE effects. The spectrum was derived from a 64 (Complex) X 32 (complex) x 1024 (real) data matrix, with acquisition times of 9.1 ms (III, t,), 17.5 ms (15N, tz), and 63.5 ms (*H, ts), respectively, and a total experiment time of 50 h. The final processed spectrum consisted of 256 x 64 x 512 real points with digital resolutions of 0.14, 0.48 and 0.05 ppm/pt in the wI, 02 and wS dimensions, respectively. The 3D {‘H,15 N} NOESY-HMQC spectrum [33,36] was acquired with solvent presaturation during both the relaxation delay and the first IOOms of the 125 ms mixing time. The acquistion and processing parameters were essentially identical to those of the TOCSY-HMQC, except only 16 complex points were collected in tZ (lsN>, reducing the acquisition time to 9.7 ms, the total time to 25 h, and the digital resolution to l.Oppm/pt. The HNCO, HNCA [lo] and HN(CO)CA [37] spectra were recorded in H,O, with carrier frequencies set at: 1f I, 4.75 ppm; 15N, 117.88 ppm; W,, 56.00ppm; WO, 176.OOppm. For the HNCO experiment, the data matrix consisted of 32 (complex) X 64 (complex) x 1024 (real) points in the t,, t2 and t3 dimensions, respectively, with acquisition times of 17.5 ms (l5N, tI), 35.3 ms (WO, t2) and 63.5 ms (‘H, t3> and 16 scans per (tl, tz> point for a total time of 40 h. The final data matrix was 64 X 256 X 1024 real points, giving a digital resolution of 0.47, 0.05 and O.O07ppm/pt in ol, w2 and 03. For the HNCA experiment, the data matrix consisted of 32 (complex) x 64 (complex) X 1024 (real) points in the ti, t2 and tj dimensions, respect tively, with acquisition times of 17.5ms (l5N, t,), 15.1 ms (W&, tz) and 63.5 ms (‘H, t3) and 32 scans per (tl, tz> point for a total time of 80 h. The final data matrix was 64 x 256 x 1024 real points, giving a digital resolution of 0.47, 0.11 and 0.007 ppm/pt in o,, w2 and 03. For the HN(CO)CA experiment, the data matrix consisted of 16 (complex) x 32 (complex) x 1024 (real) points in the tl, tZ and t3 dimensions, respectively, with acquisition times of 8.77ms (l5N, tl), 7.55ms (‘3Ca, t,) and 63.5ms (‘H, t3) and 64 scans per (t,, tz) point for a total time of 3811. Processing parameters were the same as those for the HNCA experiment. The HCACO and HCA(CO)N spectra [lo] were recorded in QO, with the same carrier frequencies used above. For the HCACO experiment, the inital data matrix was 32 (conplex) X 64 (complex) x 1024 (real) points in tl, t2 and tg, A total of eight scans were signal averaged per (tl, tz> point, with acquisition times of 6.4 ms (W,, tl), 28.3 ms (1X0, tz), and 72.3 ms (‘II,, t3) and a total experiment time of 22 h. The fiIX1 data matrix was 64 X 256 X 1024 real points, yielding digital resolutions of 0.52, 0.06 and 0.006ppm/pt in wl, w2 and oj. For the HCA(CO)N experiment, the inital data matrix was 16 (complex) X 32 (complex) X 1024 (real) points in t,, t2 and t3, Thirtytwo scans were signal averaged per (cl, tz) point using a 32 step phase cycle, with acquisition times of 6.4 ms (WCC, t,), 7.8ms (l%O, tz), and 72.3 ms (IHa, t3> and a total experiment time of 22 h. The final data matrix was 64 x 256 x 1024 real points, yielding digital resolutions of 0.52,0.06 and 0.006 ppm/pt in wl, o2 and w3, respectively.

Acknowledgements: We thank Dennis ‘Torchia and Milo Westler for their instruction, advice and encowdgement in the early stages of this work and Alan Meeker for preparing all of the prorein samples used. We thank Andrei Alexandrescu, David Weber, WeiJer Chang,

and tiberr Miki&Ill for helpful discussions. This work was supported by NIH grants GM34171 to DS and 11~28616 to Albert S Mildvan.

of a denatured

protein

Shortle

and

Abeygunawardana

References Pakula, Ah. & Sauer, R.T. ( 1989). Generic analysis of protein stability and function. Awzu. Kelp, Gencl. 23, 289-310. Shorrle, D. (1392). Mutadonal studies of protein strucmres and their stabilities. Q, Rer! Riophys 25, 205-250. Matthews, B.W. (1993). Strucrural and genetic analysis of protein stability Annu Rez! Rio&m. 62, 13940. Shortle, D. & Meeker, A.K. (1986). Mutant forms of staphy lococcdt nuclease with altered patterns of guanidine hydro~ chloride and urea dena&~ration. Proleins, 1, 81-89. Shortle, D., Stites, W.E. Sr Meeker, A.K. (1990). Contnhutions 5 of the large hydrophobic amino acids to rhe stability of staphy~ lococcal nuclease. Riochemistry, 29, X033-8041, 6. Green, SM., Meeker, A.K. 81 Shortle, 1). (1992). Contributions of the polar. uncharged amino acids to the stability of staphyIococcaI nucl~ase: evidence for mutational effects on the free enerkv of the denatured state. Biochemistry, 31, 5717-5728. Shortle, D. & Meeker, AK. (1989). Residual srructure in large 7. fragments of sraphylococcal nuclease: effects of amino acid substitutions. Biochemistry, 28, 936944. Neri, D., Wider, G. S: Wiithrich, K. (1992). Complete ljN and 8. IH NMR assignments for the amino-terminal domain of the phage 434 repressor in the urea-unfolded form. Proc. Natl. Acad Sci l/S4 89, 4397-4401. Eagan, D.A., Logan, TM., Ilang, H., Mdtayoshi, E., Fesik, S.W. 9 81 H&man, T.F. (1993). Equilibrium denalUrdtion of recombinant human FK binding protein in urea. Hiocbcmisty,32, 192(r1927. 10. Kay, L.E., Tkurd, M., Tsciiudin, K. & Hax, A. (1990). Three dimensional triple resonance NMR specrroscopy of isotopically enriched proteins. J ~!&~n. l&ox 89, 496514. 11. Ikurd, M., Kay, L.E. & Bax, A. (1990). A novel approdch for sequential assignment of IH, ‘3C, and ‘5N spectra of larger proteins: heteronuclear triple resonance three~climen sional NMR spectroscopy, Application to calmodulin. Riochernishy, 29, 46594667. D.A., Sparks, SW. 8r Bax, A. (1989), Staphylococcal 12. Tol-chia, nuclease: sequential assignments and solution stmcture. Rio cbemihy, 28, 5509-5524. U.K. 81 Forman Kay, 13. Kay, LE., Xu, G., Singer, A.U., Muhandiram, J.D. (1993). A gradient~enhanced HCCH-TOCSY experiment for recording side chain ‘H and 13C correladons in Hz0 samples. J Magn. A+sOlL. 101, 333-337. 14. Wang, J., Hinck, A.P., 1.011, S.N., L&laster, D.M Xr Mdrkky, J.L ( 1992). Solution studies of staphylococcdl nucl~dse 11124L. II. ‘H, l%Z, and ‘5N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-t~~~~lTliclille 3’,5’~bisphosphatcCa2+ ternary complex. Biochemisty, 31, 921-936. 15. Wisharr, D.S., Sykes, B.D. & Richards, FM (1991). Relationship between nuclear magnetic resonance chemical shift and prorein SecOnddry StTUCtllR!. ,I hfO[ fhO[ 222, 311~333. D.S., Sykes, B.D. & Richards, F.M. (1992). The chemi16. Wishart, cal shift index: a fast and simple method for the assignment of protein secondary srructure through NMR spectroscopy. Bio chenzistq 3 1, 1647- I65 I. 17. Spera, S. & Bax, A. (1991). Empirical correlation between protein backbone conformation and Ccc and Cp ljC nuclear magnetic resonance chemical shifts. J Am. Chcm Sot. 113, 549&5492. 18. Gower, J.C. (1971). General coefficient of similaril) and SOI~K of its properties. Biometrics, 27, 857-871. C.M. (1992). Unfolded proteins, compacr States ar~f 19. Dobson, molten globules. Czrrr. @in. Stwct Riol 2, Gl2. states of proreins and [heir t&s 20. Shortle, D. (1993). Denatured in folding and srability. Cwz Opin. Slruct. Riol. 3, 66 74. 27. Dill, K.A. (1990). Dominant forces in protein folding. ~~~C~~~misty, 29, 7133-7155. Introduction of a disuhde 22. Belz, S.F. & Pielak, G.J. (1992) bond imo cytochrome c stabilizes a compact denatured state. Biochemistry, 3 1, 12337-I 2344. of protein folding 23. Shortle, D. (1989). Probing the clerermmants and stabilily with amino acid substitutions. / /?iOL &em 264. 5315-5318.

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32.

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34.

35.

36.

37.

Marion, 11., Ikurq M. 81 ISax, A. (1989). ImpI-wed solvent suppression in one- and two~dimensional NMR spectra by convo lution of time domain data. ./. ~U’qqn. K~on. 84, 425 -430. Marion, D., cf al, 8r Clore, GM. (1989). Overcoming the overlap problem in the assignment of ‘11 NMR spectra of larger proteins by the use of three~dimensional heteronuclear ‘I I t TN I Iarttnann~Ilahn~rnllltiple quantum coherence and nu clear Overhauscr-multiple quantum coherence spectroscopy: application to intedeukin 18, Biochernbt~y, 28, 6150 6156. Pelton, J.G., ‘I‘orchia, I>.A., Mcddow, N.D., Wong, C.~Y. 6i Roseman, S. (1991). lI1, lsN, and I% NMR signal assign ment of II@, a signal transducing protein of B coli, us ing three~dimensional triple resonance techniques. l?icdwrn iktq 30, 10043- 10057. Shaka, AJ., Lee, CJ. & Pines, A. (1988) Iterative schemes of hilineal- operators: application to spin decoupling. J &‘qn. f&sort. 77, 2744238. Zuiderweg, E.R.P. 8r F&k, S.W. (1989). Heteronuclcar three dimensional NMR spectroscopy of the inflammatory protein C5a. Riochemistry, 28, 2387-2391. Uax, A. & Ikura, M. (1931). An efficient 3D NMK technique fol- corrclatmg the proton and ‘5N backbone amide reso nances with the c( carbon of the preceding residue in uniformly ‘5N/‘-‘C enriched proteins. .j Riornd NMR, I, 99-104.

Kecewcd: Accepted:

16 July 1993; 2 September

revised: 1993.

1 September

1993.