Biochimica ,,t Biophysica Acta. 1121 (1992) 8-15 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34170
Hydrogen exchange in Pseudornonas cytochrome c-551 Russell Timkovich, Larry A. Walker II and Mengli Cai Department of Chemistry. Unicersio' of Alabama. Tuscaloosa, AL (USA) Received 23 September 1901)
Key words: Hydrogen exchange: Cytochrome: NMR: (Pwudomtmo~)
Hydrogen exchange rates were measured or estimated for 75 amide protons in ferrocytochrome c-551 from P~eudomonas aeruginosa (82 residues total) at neutral pH and 300 K. Rate constants span at least eight orders of magnitude. Rate constants or limiting estimates were determined by a combination of methods relying upon ~H-NMR spectroscopy, including the direct observation in one- or two-dimensional spectra of the decrease in proton intensity for samples dissolved in deuterium oxide, or, in a few favorable cases, saturation transfer from the solvent protic water. The heme ligand residues and the thiocther bridge residues were slowly exchanging backbone amides, but the slowest exchanging backbone amides were found in two clusters. One was composed of Iic-48 and Lys-49 in the last turn of what is termed the 40's helix in the protein. The second was composed of Leu-74, Ala-75, Lys-76 and Val-78 in the C-terminal a helix.
Introduction
Materials and Methods
Investigation of hydrogen exchange kinetics is now a well recognized technique for the study of protein stability and conformational flexibility. With the advent of high resolution N M R and the powerful assignment techniques of two-dimensional spectroscopy, it has become possible to determine exchange behavior at specific sites. Illustrations are available in the literature [1,2-5] Recently, Englander, Roder and co-workers have made extensive investigations of the exchange behavior of labile protons in oxidized and reduced horse cytochrome c as well as folding intermediates [6-9]. Cytochrome c-551 is an electron transport protein found in many bacteria where it performs an analogous function to mitochondrial cytochrome c. it has fewer residues than horse cytochrome c (82 versus 104), but still demonstrates sequence and folding homology with the larger protein [10]. Because of the deletions there are structural rearrangements and these raise the question of the relative stability of the smaller protein. We have attempted to explore this issue by measuring amide hydrogen exchange rates in cytochrome c-551 from Pseudomonas aeruginosa.
Pseudomonas aeruginosa (ATCC 19429) was cultured and cytochrome c-551 was purified as described in Ref. I I. With our fermentation equipment and considering the cytochrome content of the cells, it is feasible over time to isolate c-551 in amounts of 10-100 rag. However, hundreds of milligrams are not available. This precluded the use of material-consuming methods for determination of a complete range of exchange rates as d e ~ r i b e d by others [12]. By a combination of techiniques, it was possible to measure most main chain amide exchange rates and determine limits for others. N M R samples in nominal protic water contained 10% by volume deuterium oxide to provide spectrometer lock. They were prepared by dissolving lyophylized protein in 50 mM pota~ium phosphate buffer to a final concentration, typically 5 mM in protein. The pH was adjusted to 7, and the solution deoxygenated by cycles of vacuum and argon flushing. The cytochrome was reduced to the ferrous state with minimal solid sodium dithionite, and then sealed in 5 mm N M R tubes under an argon atmosphere. The time-course of exchange was followed in 99.9% deuterium oxide where the intensities of residual amide resonances were monitored. Samples were prepared in a special way to ensure that the rates reflected the behavior of the ferrocytochrome. Protic solutions of ferrocytochrome
Correspondence: R. Timkovich, Department of Chemistry, University of Alabama. Tuscaloosa. AL 35487-0336, USA.
were lyophylized directly in the NMR tube. An equivalent volume of deuterium oxide plus a slight amount of solid sodium dithionite was added to begin the exchange. The tube was then sealed under argon. It required 16 min before the first NMR spectra could be acquired. Amide resonances were identified based upon published assignments [13-15]. Spectra were recorded at 500 MHz at 27°C. Kinetic samples were placed in a bath at this temperature when not in the regulated spectrometer probe. Some .samples were kept under controlled conditions and systematically examined for 3 months. The slow exchange rates were measured by following the decrease in proton intensity with time and fitting the decrease by linear regression to a firstorder rate law. Resolved amide resonances were monitored in one-dimensional spectra. In principle, overlapping amide resonances could b~ followed by their cross peaks in several different kinCs of two dimensional spectra. We selected H O H A H A spectroscopy [16] as described previously [13]. Lineshapes arc nearly pure absorption mode, and this improves resolution in portions of the fingerprint region, in our hands, the signal to noise ratio for the majority of needed cross peaks was higher for short acquisition times in H O H A H A spectra compared to NOESY spectra. Using 16 ~ans per t l increment and 300 increments, a spectrum with acceptable noise could be acquired in 2.25 h. The decrease in intensiW of the amide to a cross peak was followed to determine the exchange rate. There are several cases at this temperature and pH where even these types of cross peak overlap, including the pairs Ala-71/Ala-75, Lys-76/GIn-72 and Val-30/Ala-32. By using H O H A H A spectra it was then possible to follow the disappearance of" the amide proton by the relayed cross peak to ~ protons. Saturation transfer experiments in protic solutions [17] were performed to measure rates for the fastest exchanging residues. Because of the huge water resonance these were done by comparing a one-dimensional spectrum taken with a jump and return read pulse [18], versus a normal spectrum preceded by a soft, long presaturation pulse. Note that you cannot precede a jump and return read pulse by a presaturation pulse, because the success of jump and return depends on having all the water magnetization aligned along the z axis at the start, lump and return spectra do not have a uniform excitation pro0!e ~ r , - ~ the aromatic region, but it was possible ~o scale spectra together over small portions of a few tenths of a ppt~ by using nearby resonances that arc not exchangable ~,r known to be slowly exchanging frorl other data. This technique has been exploited by others [5]. For amides in calbindin subject to overlap, but with exchange rates sensitive to saturation transfer, Linse et al. reported rate estimates based upon variable presaturation times
prior to recording COSY spectra [5]. Similar experiments were attempted for c-55 l, but wc were unable to observe differences. Results
Because of the wide range of exchange rates, amide protons were placed into different classes according to how their exchange rate constants were measured. Results are summarized in Table I. Class IA represented protons that were resolved by chemical shift at any time, or were being sufficiently slowly exchanged so that they were resolved from overlapping peaks in some late time frame. Their intensity could be followed as an indivdiual site and the decrease was fit by regression analysis to a simple firstorder decay. Fig. 1 demonstrates that there were several such protons. Class IB is like IA except too few data points were captured for a meaningful statistical estimate of error. Class I I represented protons that were not resolved, but their exchange rate was much faster than overlapping peaks and could be measured from the fast phase of intensity decrease in one-dimensional spectra. Fig. 2 illustrates how this was possible for the case of Lys-33 and Trp-77. Class IliA represented protons that were not shiftresolved in one-dimensional spectra in any time frame. Rates were measured by the decrease in cross peak intensity in two-dimensional HOHAHA spectra. Class IIIB is like IlIA except that only a few data points were captured. Class IVA represented protons at least partially resolved in one-dimensional spectra that had no detectable ~turation transfer effect from the solvent. They are fast exchanging protons because they were absent by the first one-dimensional spectrum recorded in 2H20. A limit was placed on their rate constant by the following considerations. By the first 2H-spectrum they appeared to have lost at least 90% of their intensity. So it was assumed that at least three half-lives had passed during the 16 min deadtime required to record the first spectrum. So the half-time for exchange must be less than approx. 5 min, or the first-order rate constant greater than about 0.1 min -~. Class IVB represented protons not resolved in one-dimensional spectra, but whose cross peaks were absent by the first H O H A H A spectrum recorded in 2H20. The ratc constant estimate was made as described for class IVA considering the deadtime for the first two-dimensional spectrum. Class IVC represented protons that satisfied the conditions for class IVB, but demonstrated an additional property. They gave rise to intense chemical exchange cross peaks with the solvent in H O H A H A spectra recorded in protic water. This is illustrated in
10 TABLE !
Soh'ent exchange rate constants flor P. aeruginosa ~errea'ytochrome ,'.551 at 27°C ana p H 7 ,.
Residue Glu-1 Asp-2 Pro-3 Glu-4 Val-5 Leu-6 Phe-7 Lys-8 Asn-9 Lys- I 0 GIy- I ! Cys- 12 Val- 13 Ala- 14 Cys-15 His-16 His-16P ~ Ala-17 lie-18 Asp-19 Thr-20 Lys-21 Met-22 Val-23 Gly-24
Shift ~
k,.~ (rain - I)
D a t a class h
n.d. 9.43
6"IO-"
VII VB
8.30 7.02 7.54 8.39 6.91 8.81 8.99 7.90 8.49 6.611 7.34 6.9{I 6.77 8.68 7.72 8.32 8.02 6.48 8.44 7.32 6.93 6.65
> I0- 2 6.8(0.8)" I0- z 5.7(0.5)- 10- 4 n.d. 6.5(0.6)- 10 - 3 > 10 --~ 1.15(0.02). I0 -~ >I0--" 7.2(0.2)- I 0 - 4 3.4(0.1 )- 10 3 9.10- ~ 5.3(0.4)- I 0 - 4 4.6(0.2). 1 0 ~ 6.1(0.4)-I0 -4 3.2(I}.6)- I 0 - 4 > 10 -2 >i0-~ > I 0 -I 5.6 I(0.07)- I0 -? > 10 -a > I0-" 1.52(0.04)- I 0 - 4
IVB IA IA Vii IliA IVB IA IVB IA IA lllb IliA IA IA Ilia IVC IVA IVA IA IVB IVB IA
3.8(0.1 ). 10 .-3 n.d. >10 -t !.3(0.1). 10 -3 8(2)" I0 - c, 1.4(0.3). 10 - s 8(2)- I 0 - s 1.5(0.2)-I0 ~ 4.2(0.3). I0 - ~ n.d. 4.6(0.2). I0-2 > I0 -2 4' 10 2 4" 10 2 > 10 .2 > I0 -t > I0 -2 > i0 --~ 7.7(0.6)- 10- ~ 5.0(0. I). 10 - "~ 1.2(0.2). I0--" 5(I). I0 -~ > 10 -2
I! Vll IVA !1 IliA !!
Pro-~ Ala-26 Tyr-27 Lys-28 Asp-29 Val-30 Ala-31 Ala-32 Lys-33 Phe-34 Ala-35 Gly-36 Gin-37 Ala+38 Gly-39 Ala-40 Glu-41 Ala-42 Glu-43 Leu-44 Ala-45
Gin-46 Arg-47 Arg-47E d Iie-48 Lys-49 Asn-50 Gly-51 Ser-52 Gin-53 Gly-54 Val-55 Trp-56 T~-561 " Gly-57 Pro-58
8.47 7.54 8.89 6.77 7.59 8.62 7.59 7.81 7.71 7.68 8.90 7.92 8.96 9.(19 7.65 8.87 7.82 7.75 8.63 8.31 7.35 7.64 7.5 I 8.15 7.14 8.09 7.4 I 7.19 7.93 8.41 10.46 10.76 11.90 7.99
< 1 0 -~ < 1 0 -7 1.82(0.03). I0 - 4 2.2(0. I ). 10 ~ 2.70(0.07)- 10- 4 > 10 --~ > 10 -2 4.7(03)-10 -z i.78(0.02). 1 0 3 2.10 -t 7.10-2
Ilia II III Vll IA IVB VA VA IVC IVA IVC IV(? IA IA IA Ilia IVB VIB VIB IA IA IA IVC IVC IA IA 1B IB
11 T A B L E 1 (continued) =
Residue
Shift "
/%, (rain 71)
Data class h J ,
Ile-59
7.49
>10-2
Pro-60
-
-
Met-61 Pro-62 Pro-63 Asn-64 Ala-b5 Val-66 S¢r-67 Asp-68 Asp-69 Glu-70 Ala-71 Gin-72 Thr-73 Leu-74 Ala-75 Lys-76 Trp-77 Trp-771
8.74 6.96 8.79 7.70 9.26 8.91 8.43 7.88 8.83 8.14 8,25 8.61 8.83 8.19 7.82 111.21
Val-78
9.21
Leu-79
7.76
Ser-80 Gin-81 Lys-82
7.46 7.16 7_52
-
!.4(0.41-Ill
IVB IA
"
7.7(0.21- I0 5 > Ill - 2 2.2. Ill 2 5(0.91.10 - : 4-10-" > ll}- 2 > 10 : 2.4(0.2)-I(I 4 7.7(0.5)- l0 -4 1.65(0,03). I(1 2 < !11 ? l-I{I -r 4.5(t).6)- I t ) - " 4.2(0.5)-l(| ~ > 1O ] < 10 - 7 I0 2 > 10 z
iliA IVC IlIB IA VB IVC IVB !i IliA IA VIB VIA IliA IA IVA VIB
VIB IA IVB IVC
a Chemical shift in ppm for the proton. Shifts were determined from H O H A H A spectra recorded in the present study, but are in substantial a g r e e m e n t with published assignments in Refs. 13-15. t, Data classes represent how the rate constant or estimate was obtained as discu.~sed in the text. The number in parentheses for a rate constant represent the statistical standard deviation from the regression analysis. c T h e side chain imidazole pi proton. The other nitrogen d o n a t e s a lone pair of electrons to the heine iron for one ligand I~md. d The side chain epsilon proton of Arg-47. ': The side chain indole proton.
~-
,~'.~
,;.o
d.s
Io'.o
9:~
e |5 9.0 PPM
olo
715
71o
615
6.0
Fig. I. The aromatic region of P. aeruginosa ferrocytochrome c-551 containing exchangeable amide remmances. The botlom spectrum was taken in protic water at pH 7 with a solvent presaturalion pulse. The bottom spectrum was taken 10 h after transferring a sample to deuterium oxide.
12 I
r'
i
l 4.000
,, 6000
i
This type of cross peak also appears in NOESY spectra (with the same pha~ as regular NOESY cross peaks) that incorporate a selective excitation pulse sequence for the final read pulse (e.g., [22]), but are attenuated or absent in NOESY spectra with water presaturation. The presence of these exchange cross peaks indicates that the halftime for exchange is likely on the order of the duration of the spinlock pulse in the H O H A H A sequence, or a rate constant approaching 102 rain- ~ or even higher. These are clearly special cases that occupy a distinctive time window for exchange. Note in Fig. 3 that amides resolved in one-dimensional spectra that show a saturation transfer effect also show H O H A H A exchange cross peaks. Table ! reports a conservative estimate for the rate constant of IVC amides, based upon the criterion of class IVB, because at the present time we cannot quantitatively evaluate the meaning of the magnitude of the exchange cross peak. A discussion of the problems of quantitating exchange cross peaks has been given in a recent review by Pert'in and Dwyer [23]. Class V r e p r e ~ n t e d protons that showed saturation tran~;fer effects with water. The saturation transfer experiments had only limited success. The half-lives of some amides did not fall into the range accessible by saturation transfer. Another problem was posed by a proton resonances under or very near to the broad solvent resonance. These a protons are close in spatial distance to some target amides. Even using low power, selective presaturation pulses on water unavoidably excites the a proton resonances, and since the time scale is the same for dipolar magnetization transfer, negative nuclear Overhauser enhancements occur. These cause intensity decreascs that are indistinguishable from saturation transfer. Data class VA represented amides that were sufficiently resolved for obvious, strong saturation transfer
1.6
1.4
Lys 3;~
1.2
0.8 0.6 ' 0
i
, 2000
"~__8000
.time (min.) Fig. 2. First-order ph)t of the logarithm of remaining intensity (arbitrary units) versus time fi)r two amide protons that extensively overlap in chemical shift. The solid lines are the regression analysis [it for the exchange rate constant of the fast phase (Lys-32) and the slow phase (Trp-77). The intensity measurements were made in one-dimensional spectra, but the identification of the respective fast and slow phases was based upon cross peaks in two-dimensional spectra.
Fig. 3. The peaks have the same phase as cross peaks for scalar correlated protons, opposite to the phase of rotating frame NOE peaks, and lie at the chemical shift of the amide and the shift of water [19,20]. Sophisticated methods exist to discriminate these exchange cross peaks from true scalar correlation peaks [21], but in practice they are easy to identify if one records spectra at several different temperatures. The water resonance is usually much more temperaturesensitive than a proton resonances, and the exchange cross peaks always move in a band with the water peak.
.
9
G54=
0
0
K28o~
0
L74='
A65o~ $67~
0
D2X.D2=~
4.6 G39X
@
©)
A38X D68X~TA65X
D69X
o
4.8 G54X CI2¢x
!
T
9.4
!
9.3
4.4
D69¢~
9:2
T 9.1
"l 9.0
PPN
o'.9
81o
| 0.7
.:s
t 8.5
5.0
7 Pl~ 9.4
Fig. 3. A lx)rtion of the two-dimensional H O H A H A spectrum of P. aeruginosa c'-551. Cross peaks representing scalar coupling between the amide proton and the or proton are marked wtih the one letter code for the residue. Chemical exchange cross peaks with the water are marked with an X. They distinctively lie in a band at the water chemical shift in the FI dimension in this quadrant of data. The situation with Asp-2 illu.~trates how an a proton can overlap with the water re,~)nance.
13 to be observed, and, from our knowledge of assignments and NOESY spectra, there was no opportunity for an NOE involving an t~ proton under or near the water peak. Class VB represented amides that showed a marked decrease in intensity in saturation transfer experiments, but the magnitude is ambiguous because of the potential for an N O E involving an a proton. The rates reported assume that the observed decrease is solely due to saturation transfer. If there were a major NOE, for example say a 50% negative enhancement, then the effect would bc that the true exchange rate constant is 10-times smaller. Significant intensity changes were observed in saturation transfer experiments around 8.4, 7.9 and 7.5 ppm, but these could not be interpreted because of severe overlap and ambiguity involving NOE effects. There are class IVC protons in each of these ranges. Class VI represented the most slowly exchanging amides. A pH 7 sample sealed under argon had been kept for over 2 years at room temperature. It was not systematically studied for this time, but it was possible to measure the remaining intensity of the extremely slowly exchanging protons and perform a two point (times zero and 2 years) estimate of the rate. Class VIA protons had intensities of less than one, while VIB protons had intensities still within experimental error of one proton, and hence for practical purposes are non-exchangable. Class VII represented four protons for which not even approximate estimates could be made. This included the free amino terminus of Glu-I for which no assignment has been made. Phe-7, Tyr-27 and Ala-35 have been assigned, but they are in crowded regions in one-dimensional spectra. In H O H A H A spectra in ] H 2 0 ( n o exchange kinetics) they give weak cross -8
i
i
10
20
peaks because of unfavorable coupling constants between the amide and a protons. Based upon the crystal coordinates for c-551 the predicted constants are 1.7 (Phe-7), 3.0 (Tyr-27) and 6.2 Hz (Ala-35). The crystaI predicted 6.2 Hz value for Ala-35 is too high. A recent solution structure for P. aeruginosa c-551 based upon scalar and NOE constraints [24] suggests extensive differences between crystal and solution around residue 35 and our own COSY spectra indicate a small coupling constant, less than 4 Hz, for Ala-35. The H O H A H A cross peaks thus start out with an initial intensity that is low and were absent by the first H O H A H A spectra in ZH solvent. This is at first glanc~ like a class IVB proton, but the starting intensity was so weak that they could be exchanging slower and the residual cross peak intensity was obscured by noise. Side chain amide protons have been assigned in P. aeruginosa c-551. In general they are in crowded spectral regions where there are other rapidly exchanging protons. They arc observable in H O H A H A spectra in the aromatic region (see Fig. 3, Ref. 25) and are absent by the first kinetic two-dimensional data point. They could belong to class IVB or V, but there is insufficient data to so place them. They are not in any slow class. Discussion
The rates of exchange are presented in a graphical form in Fig. 4. Class VIIB protons for Fig. 4 were arbitrarily assigned a log k value of -7.5 to place them on the graph, but are specially marked. Class IVC protons were arbitrarily assigned a value of 2 for the same reason and are also specially marked. Other amides that have not been quantified or for which only an estimate has been made are omitted. However, bear
i
,
30
40
-6
$ -2
4.
i 0
residue
I
I
I
I
50
60
70
80
number
Fig. 4. Histogram representing the dislribulion of exchange rates in P. aen~ginosa c-551. Data have been takc~ .¢rom Table I. The open bars represent residues where a single valued measurement has been made. The solid bars represent the .slowest exchanging residues where only a limiting estimate of less than 10 -7 rain- 1 has been made. These were arbitrarily assigned a value of - 7 . 5 for display purpt~ses. The cross-hatched bars represent residues that show chemical exchange cross peaks in H O H A H A spectra. Although only a limit of greater than 10 z rain - ~ has been assigned to these nlles in Table I. they are depicted here with an arbitrary value of 2 for display purposes.
14 in mind that these arc amides that exchange too slowly for saturation transfer or two-dimensional chemical exchange effects, but are fast enuogh to be gone by the first spectra in "H-solvent. it is likely that their log k values are between - 1 and 1. A stereoview of c-551 is given in Fig. 5. Some of the present data can be compared to exchange rates determined for horse heart cytochrome c [6-9]. Cyt c is a larger protein, 104 residues, but it shares many features with c-551, especially the ligand geometry, and N- and C-terminal a helices. The N- and C-terminal residues exchange rapidly and this is perhaps to be expected. A segment from residues 35 to 43 shows consistently high exchange rates. The crystal structure of c-551 [I0] reported a type I turn from residue 34 to 37 and a type I1 turn from 37 to 40. Residues 41-43 mark the beginning of what was termed the 40's helix (41-50). In the crystal structure determination, this segment had some of the highest diffraction mean temperature factors in the entire protein (see especially Fig. 6 of Ref. 10). High temperature factors could represent either static disorder in the crystal or large mean amplitudes of thermal motion. The crystallographic data and the exchange data both mark this region as special in the structure of the protein. Residues 18-20 are in another fl bend on the protein surface and this could rationalize the relatively high exchange rates here. They also have above normal temperature factors in the crystal structure, but not as high as around residue 36. Residues 67-70 are at the very beginning of the C-terminal helix and are at least partially solvent exposed in the crystal structure. Once again Asp-68 has an above average temperature factor.
The slowest exchanging amides can be rationalized in broad terms by hydrogen bonding and solvent inaccessibility. The quantitative exchange data is consistent with the hydrogen bonding pattern for helix geometry (carbonyI of the nth residue to the amide of n + 4). However, the actual relative rates could not have been predicted. In the previous paragraph it was shown that very fast exchange rates correlated well with high mean temperature factors in the diffraction data. The correlation does not work for the very slow residues. They have a factor of approx. 10 A 2, which is about the average or median value. The heme ligands and residues around the thioether bonds are slowly exchanging. This has also been noted for cytochrome c. The Met-61 heine ligand appears distinctive in Fig. 4, but it is surrounded by proline residues at 58, 60, 62 and 63. Slow exchange pertains for ot helical segments, especially the helices from residues 27 through 34, the 40's helix from 40 through 51 and the C-terminal helix from 70 through 80. It was unexpected that the very slowest exchanging residues in c-551 were at the very ends of the 40's helix and C-terminal helix. There is nothing obvious in the crystal structure that earmarks these particular residues. Compare, for example, Leu44, a residue in the middle of the helix that orients its side chain toward the protein interior, with lle-48, which could be described in a similar way, and Lys-49, whose side chain extends into solution. The latter two residues have exchange rates 4-5 orders of magnitude slower than the former. It is possible that for unclear reasons, the ends of these two helices have especially stable hydrogen bonds. These slow residues have rates comparable to the slowest in horse cytochrome c and demonstrate that equivalent nonlabile amides can exist
Fig. 5. Stereoview of P. aeruginosa c-551 based upon the crystal coordinates of Re[. 10. The main chain atoms and carbonyl oxygen are shown to depict the folding paltern. The heine, heine ligands and thioelher bridges are shown to orient the active site. The N-terminal residue is at the top back and the N-terminal helix runs toward Ihe viewer. The 27-34 helix is visible on the lower right side and the 40's helix on the left side. The C-lerminal helix is at the back of Ihis view and runs diagonally a c r e s the view [rom the upper left to the lower right.
15
even in the smaller cytochrome. The end of the Cterminal helix is also the slowest segment in oxidized horse cytochrome c [9]. The 27-34 helix and the 40-50 helix in c-551 have no directly homologous elements in cytochrome c (ar~ informative comparison is Fig. 3 in Ref. 26) The 40's helix is loosely similar to a helix region in cytochrome c from 62-69, but the orientation and packing against the heme are different. Both cytochrome c and c-551 have an N-terminal a helix. In horse cytochrome c this is a region of very slow exchange 10 -~ to 10 -~ min-m for residues lie-9 and Phe-10 [6]. In c-551 it is not exceptionally slow, but moderate at around 10 -3 rain -~. The helix is inherently shorter in c-551 (a possible 12 residues till the first thioether versus 14 in cytochrome c) and Pro-3 shortens the possible length even further. This suggests greater flexibility in this region in the smaller protein. To summarize, we would like to stress the general conclusion that, in so far as protein stability is reflected by amide exchange rates, cytochrome c-551 is very comparable both overall and in specfics, to mitochondrial cytochrome c. Hypotheses that address the structure and function of c-type cytochromes should also address the fact that c-551 can achieve with circa 80 residues about the same thing as mitochondrial cytochrome c with over 100 residues. Acknowledgement This work was supported in part by grant C-~43292 from the National Institutes of Health. References 1 Tuchen, E. and Woodward, C.K. (1985) J. Mol. Biol. 185, 405-419. 2 Roder, H. and Wuthrich, K. (1986) Proteins !, 34-42.
3 Oiwen, W., Kline, A.D. and Wuthrich, K. (1987) Biochemistry 26. 6488-6493. 4 Udgaonkar, J.B. and Baldwin, R.L. (1988) Nature 335, 694-699. 5 Linse, S., Teleman, O. and Drakenberg, T. (1990) Biochemistry 29, 5925-5934. 6 Wand, A.J., Roder, H. and Englander, S.W. (1986) Biochemistry 25, 1107-1114. 7 Englander, J.j., Englander, S.W., Louic, G., Roder, H., Tran, T. and Wand, AJ. (1988)in Structure and Expression (Sarma, R.H. and Sarma, M.H., eds.), Vol. 1, pp. 107-117, Adenine Press. 8 Roder, H., EIove, G.A. and Englander, S.W. (1988) Nature 335, 700-704. 9 Jeng, M.F., Englander, S.W., EIoveo G.A., Wand, AJ. and Roder, H. (1990) Biochemistry 29, 10433-10437. 10 Matsuura, Y., Takano, T. and Dickerson, R.E. (1982) J. Mol. Biol. 156, 389-409. I I Timkovich, R., Dhesi, R., Martinkus, K.M., Robinson, M.K. and Rea, T. (1982) Arch. Biochem. Biophys. 215, 45-58. 12 Roder, H. (I989) Methods Enz~nol. 176A, 446-473K. 13 Chau, M.H., Cai. M. and Timkovich, R. (1990) Biochemistry 29, 5076-5087. 14 Detlefsen, DI., Thanabal, V., Pecoraro V.L. and Wagner, G. (1990) Biochemistry 29, 9377-9386. 15 Cai, M. and Timkovich, R. (1991) Biochem. Biophys. Res. Commun. 178, 309-314. 16 [Lax, A. and Davis, D.G. (1985) J. Magn. Re.c~n. 65, 355-360. 17 Pitner, T.P., Glickson, I.D., Dadok, J. and Marshall, G.R. (1974) Nature 250, 582-584. 18 Plateau, P. and Gueron, M. (1982) J. Am. Chem. Soc. 104, 7310-7311p. 19 Davis, D.G. and Bax, A. (1985) J. Magn. Reson. 64, 533-535. 20 Bay,, A., Sklenar, V., CIore, G.M. and Gronenborn, A. (1987) J. Am. Chem. Soc. 109, 6511-6513z. 21 Fejzo, J., Wcstler, W.M., Macura, S. and Markley, J. (1990) J. Am. Chem. Soc. 112, 2574-25771. 22 Sklenar, V. and Bax, A. (1987) J. Magn. Reson. 74, 469-479. 23 Perrin, C.L. and D ~ e r , T.]. (1990) Chem. Rev. 90, 935-967. 24 Detlefsen, D.J., Thanabal, V., Pecoraro, V.L. and Wagner, G. ( 1991 ) Biochemistry 30, 9{M0-9046. 25 Timkovich, R. (1990) Biochemistry 29, 7773-77801. 26 AImassy, R.J. and Dickerson, R.E. (1978) Proc. Nat. Acad. Sci. USA 75, 2674-2678.
BBAPRO 34170
Hydrogen exchange in Pseudornonas cytochrome c-551 Russell Timkovich, Larry A. Walker II and Mengli Cai Department of Chemistry. Unicersio' of Alabama. Tuscaloosa, AL (USA) Received 23 September 1901)
Key words: Hydrogen exchange: Cytochrome: NMR: (Pwudomtmo~)
Hydrogen exchange rates were measured or estimated for 75 amide protons in ferrocytochrome c-551 from P~eudomonas aeruginosa (82 residues total) at neutral pH and 300 K. Rate constants span at least eight orders of magnitude. Rate constants or limiting estimates were determined by a combination of methods relying upon ~H-NMR spectroscopy, including the direct observation in one- or two-dimensional spectra of the decrease in proton intensity for samples dissolved in deuterium oxide, or, in a few favorable cases, saturation transfer from the solvent protic water. The heme ligand residues and the thiocther bridge residues were slowly exchanging backbone amides, but the slowest exchanging backbone amides were found in two clusters. One was composed of Iic-48 and Lys-49 in the last turn of what is termed the 40's helix in the protein. The second was composed of Leu-74, Ala-75, Lys-76 and Val-78 in the C-terminal a helix.
Introduction
Materials and Methods
Investigation of hydrogen exchange kinetics is now a well recognized technique for the study of protein stability and conformational flexibility. With the advent of high resolution N M R and the powerful assignment techniques of two-dimensional spectroscopy, it has become possible to determine exchange behavior at specific sites. Illustrations are available in the literature [1,2-5] Recently, Englander, Roder and co-workers have made extensive investigations of the exchange behavior of labile protons in oxidized and reduced horse cytochrome c as well as folding intermediates [6-9]. Cytochrome c-551 is an electron transport protein found in many bacteria where it performs an analogous function to mitochondrial cytochrome c. it has fewer residues than horse cytochrome c (82 versus 104), but still demonstrates sequence and folding homology with the larger protein [10]. Because of the deletions there are structural rearrangements and these raise the question of the relative stability of the smaller protein. We have attempted to explore this issue by measuring amide hydrogen exchange rates in cytochrome c-551 from Pseudomonas aeruginosa.
Pseudomonas aeruginosa (ATCC 19429) was cultured and cytochrome c-551 was purified as described in Ref. I I. With our fermentation equipment and considering the cytochrome content of the cells, it is feasible over time to isolate c-551 in amounts of 10-100 rag. However, hundreds of milligrams are not available. This precluded the use of material-consuming methods for determination of a complete range of exchange rates as d e ~ r i b e d by others [12]. By a combination of techiniques, it was possible to measure most main chain amide exchange rates and determine limits for others. N M R samples in nominal protic water contained 10% by volume deuterium oxide to provide spectrometer lock. They were prepared by dissolving lyophylized protein in 50 mM pota~ium phosphate buffer to a final concentration, typically 5 mM in protein. The pH was adjusted to 7, and the solution deoxygenated by cycles of vacuum and argon flushing. The cytochrome was reduced to the ferrous state with minimal solid sodium dithionite, and then sealed in 5 mm N M R tubes under an argon atmosphere. The time-course of exchange was followed in 99.9% deuterium oxide where the intensities of residual amide resonances were monitored. Samples were prepared in a special way to ensure that the rates reflected the behavior of the ferrocytochrome. Protic solutions of ferrocytochrome
Correspondence: R. Timkovich, Department of Chemistry, University of Alabama. Tuscaloosa. AL 35487-0336, USA.
were lyophylized directly in the NMR tube. An equivalent volume of deuterium oxide plus a slight amount of solid sodium dithionite was added to begin the exchange. The tube was then sealed under argon. It required 16 min before the first NMR spectra could be acquired. Amide resonances were identified based upon published assignments [13-15]. Spectra were recorded at 500 MHz at 27°C. Kinetic samples were placed in a bath at this temperature when not in the regulated spectrometer probe. Some .samples were kept under controlled conditions and systematically examined for 3 months. The slow exchange rates were measured by following the decrease in proton intensity with time and fitting the decrease by linear regression to a firstorder rate law. Resolved amide resonances were monitored in one-dimensional spectra. In principle, overlapping amide resonances could b~ followed by their cross peaks in several different kinCs of two dimensional spectra. We selected H O H A H A spectroscopy [16] as described previously [13]. Lineshapes arc nearly pure absorption mode, and this improves resolution in portions of the fingerprint region, in our hands, the signal to noise ratio for the majority of needed cross peaks was higher for short acquisition times in H O H A H A spectra compared to NOESY spectra. Using 16 ~ans per t l increment and 300 increments, a spectrum with acceptable noise could be acquired in 2.25 h. The decrease in intensiW of the amide to a cross peak was followed to determine the exchange rate. There are several cases at this temperature and pH where even these types of cross peak overlap, including the pairs Ala-71/Ala-75, Lys-76/GIn-72 and Val-30/Ala-32. By using H O H A H A spectra it was then possible to follow the disappearance of" the amide proton by the relayed cross peak to ~ protons. Saturation transfer experiments in protic solutions [17] were performed to measure rates for the fastest exchanging residues. Because of the huge water resonance these were done by comparing a one-dimensional spectrum taken with a jump and return read pulse [18], versus a normal spectrum preceded by a soft, long presaturation pulse. Note that you cannot precede a jump and return read pulse by a presaturation pulse, because the success of jump and return depends on having all the water magnetization aligned along the z axis at the start, lump and return spectra do not have a uniform excitation pro0!e ~ r , - ~ the aromatic region, but it was possible ~o scale spectra together over small portions of a few tenths of a ppt~ by using nearby resonances that arc not exchangable ~,r known to be slowly exchanging frorl other data. This technique has been exploited by others [5]. For amides in calbindin subject to overlap, but with exchange rates sensitive to saturation transfer, Linse et al. reported rate estimates based upon variable presaturation times
prior to recording COSY spectra [5]. Similar experiments were attempted for c-55 l, but wc were unable to observe differences. Results
Because of the wide range of exchange rates, amide protons were placed into different classes according to how their exchange rate constants were measured. Results are summarized in Table I. Class IA represented protons that were resolved by chemical shift at any time, or were being sufficiently slowly exchanged so that they were resolved from overlapping peaks in some late time frame. Their intensity could be followed as an indivdiual site and the decrease was fit by regression analysis to a simple firstorder decay. Fig. 1 demonstrates that there were several such protons. Class IB is like IA except too few data points were captured for a meaningful statistical estimate of error. Class I I represented protons that were not resolved, but their exchange rate was much faster than overlapping peaks and could be measured from the fast phase of intensity decrease in one-dimensional spectra. Fig. 2 illustrates how this was possible for the case of Lys-33 and Trp-77. Class IliA represented protons that were not shiftresolved in one-dimensional spectra in any time frame. Rates were measured by the decrease in cross peak intensity in two-dimensional HOHAHA spectra. Class IIIB is like IlIA except that only a few data points were captured. Class IVA represented protons at least partially resolved in one-dimensional spectra that had no detectable ~turation transfer effect from the solvent. They are fast exchanging protons because they were absent by the first one-dimensional spectrum recorded in 2H20. A limit was placed on their rate constant by the following considerations. By the first 2H-spectrum they appeared to have lost at least 90% of their intensity. So it was assumed that at least three half-lives had passed during the 16 min deadtime required to record the first spectrum. So the half-time for exchange must be less than approx. 5 min, or the first-order rate constant greater than about 0.1 min -~. Class IVB represented protons not resolved in one-dimensional spectra, but whose cross peaks were absent by the first H O H A H A spectrum recorded in 2H20. The ratc constant estimate was made as described for class IVA considering the deadtime for the first two-dimensional spectrum. Class IVC represented protons that satisfied the conditions for class IVB, but demonstrated an additional property. They gave rise to intense chemical exchange cross peaks with the solvent in H O H A H A spectra recorded in protic water. This is illustrated in
10 TABLE !
Soh'ent exchange rate constants flor P. aeruginosa ~errea'ytochrome ,'.551 at 27°C ana p H 7 ,.
Residue Glu-1 Asp-2 Pro-3 Glu-4 Val-5 Leu-6 Phe-7 Lys-8 Asn-9 Lys- I 0 GIy- I ! Cys- 12 Val- 13 Ala- 14 Cys-15 His-16 His-16P ~ Ala-17 lie-18 Asp-19 Thr-20 Lys-21 Met-22 Val-23 Gly-24
Shift ~
k,.~ (rain - I)
D a t a class h
n.d. 9.43
6"IO-"
VII VB
8.30 7.02 7.54 8.39 6.91 8.81 8.99 7.90 8.49 6.611 7.34 6.9{I 6.77 8.68 7.72 8.32 8.02 6.48 8.44 7.32 6.93 6.65
> I0- 2 6.8(0.8)" I0- z 5.7(0.5)- 10- 4 n.d. 6.5(0.6)- 10 - 3 > 10 --~ 1.15(0.02). I0 -~ >I0--" 7.2(0.2)- I 0 - 4 3.4(0.1 )- 10 3 9.10- ~ 5.3(0.4)- I 0 - 4 4.6(0.2). 1 0 ~ 6.1(0.4)-I0 -4 3.2(I}.6)- I 0 - 4 > 10 -2 >i0-~ > I 0 -I 5.6 I(0.07)- I0 -? > 10 -a > I0-" 1.52(0.04)- I 0 - 4
IVB IA IA Vii IliA IVB IA IVB IA IA lllb IliA IA IA Ilia IVC IVA IVA IA IVB IVB IA
3.8(0.1 ). 10 .-3 n.d. >10 -t !.3(0.1). 10 -3 8(2)" I0 - c, 1.4(0.3). 10 - s 8(2)- I 0 - s 1.5(0.2)-I0 ~ 4.2(0.3). I0 - ~ n.d. 4.6(0.2). I0-2 > I0 -2 4' 10 2 4" 10 2 > 10 .2 > I0 -t > I0 -2 > i0 --~ 7.7(0.6)- 10- ~ 5.0(0. I). 10 - "~ 1.2(0.2). I0--" 5(I). I0 -~ > 10 -2
I! Vll IVA !1 IliA !!
Pro-~ Ala-26 Tyr-27 Lys-28 Asp-29 Val-30 Ala-31 Ala-32 Lys-33 Phe-34 Ala-35 Gly-36 Gin-37 Ala+38 Gly-39 Ala-40 Glu-41 Ala-42 Glu-43 Leu-44 Ala-45
Gin-46 Arg-47 Arg-47E d Iie-48 Lys-49 Asn-50 Gly-51 Ser-52 Gin-53 Gly-54 Val-55 Trp-56 T~-561 " Gly-57 Pro-58
8.47 7.54 8.89 6.77 7.59 8.62 7.59 7.81 7.71 7.68 8.90 7.92 8.96 9.(19 7.65 8.87 7.82 7.75 8.63 8.31 7.35 7.64 7.5 I 8.15 7.14 8.09 7.4 I 7.19 7.93 8.41 10.46 10.76 11.90 7.99
< 1 0 -~ < 1 0 -7 1.82(0.03). I0 - 4 2.2(0. I ). 10 ~ 2.70(0.07)- 10- 4 > 10 --~ > 10 -2 4.7(03)-10 -z i.78(0.02). 1 0 3 2.10 -t 7.10-2
Ilia II III Vll IA IVB VA VA IVC IVA IVC IV(? IA IA IA Ilia IVB VIB VIB IA IA IA IVC IVC IA IA 1B IB
11 T A B L E 1 (continued) =
Residue
Shift "
/%, (rain 71)
Data class h J ,
Ile-59
7.49
>10-2
Pro-60
-
-
Met-61 Pro-62 Pro-63 Asn-64 Ala-b5 Val-66 S¢r-67 Asp-68 Asp-69 Glu-70 Ala-71 Gin-72 Thr-73 Leu-74 Ala-75 Lys-76 Trp-77 Trp-771
8.74 6.96 8.79 7.70 9.26 8.91 8.43 7.88 8.83 8.14 8,25 8.61 8.83 8.19 7.82 111.21
Val-78
9.21
Leu-79
7.76
Ser-80 Gin-81 Lys-82
7.46 7.16 7_52
-
!.4(0.41-Ill
IVB IA
"
7.7(0.21- I0 5 > Ill - 2 2.2. Ill 2 5(0.91.10 - : 4-10-" > ll}- 2 > 10 : 2.4(0.2)-I(I 4 7.7(0.5)- l0 -4 1.65(0,03). I(1 2 < !11 ? l-I{I -r 4.5(t).6)- I t ) - " 4.2(0.5)-l(| ~ > 1O ] < 10 - 7 I0 2 > 10 z
iliA IVC IlIB IA VB IVC IVB !i IliA IA VIB VIA IliA IA IVA VIB
VIB IA IVB IVC
a Chemical shift in ppm for the proton. Shifts were determined from H O H A H A spectra recorded in the present study, but are in substantial a g r e e m e n t with published assignments in Refs. 13-15. t, Data classes represent how the rate constant or estimate was obtained as discu.~sed in the text. The number in parentheses for a rate constant represent the statistical standard deviation from the regression analysis. c T h e side chain imidazole pi proton. The other nitrogen d o n a t e s a lone pair of electrons to the heine iron for one ligand I~md. d The side chain epsilon proton of Arg-47. ': The side chain indole proton.
~-
,~'.~
,;.o
d.s
Io'.o
9:~
e |5 9.0 PPM
olo
715
71o
615
6.0
Fig. I. The aromatic region of P. aeruginosa ferrocytochrome c-551 containing exchangeable amide remmances. The botlom spectrum was taken in protic water at pH 7 with a solvent presaturalion pulse. The bottom spectrum was taken 10 h after transferring a sample to deuterium oxide.
12 I
r'
i
l 4.000
,, 6000
i
This type of cross peak also appears in NOESY spectra (with the same pha~ as regular NOESY cross peaks) that incorporate a selective excitation pulse sequence for the final read pulse (e.g., [22]), but are attenuated or absent in NOESY spectra with water presaturation. The presence of these exchange cross peaks indicates that the halftime for exchange is likely on the order of the duration of the spinlock pulse in the H O H A H A sequence, or a rate constant approaching 102 rain- ~ or even higher. These are clearly special cases that occupy a distinctive time window for exchange. Note in Fig. 3 that amides resolved in one-dimensional spectra that show a saturation transfer effect also show H O H A H A exchange cross peaks. Table ! reports a conservative estimate for the rate constant of IVC amides, based upon the criterion of class IVB, because at the present time we cannot quantitatively evaluate the meaning of the magnitude of the exchange cross peak. A discussion of the problems of quantitating exchange cross peaks has been given in a recent review by Pert'in and Dwyer [23]. Class V r e p r e ~ n t e d protons that showed saturation tran~;fer effects with water. The saturation transfer experiments had only limited success. The half-lives of some amides did not fall into the range accessible by saturation transfer. Another problem was posed by a proton resonances under or very near to the broad solvent resonance. These a protons are close in spatial distance to some target amides. Even using low power, selective presaturation pulses on water unavoidably excites the a proton resonances, and since the time scale is the same for dipolar magnetization transfer, negative nuclear Overhauser enhancements occur. These cause intensity decreascs that are indistinguishable from saturation transfer. Data class VA represented amides that were sufficiently resolved for obvious, strong saturation transfer
1.6
1.4
Lys 3;~
1.2
0.8 0.6 ' 0
i
, 2000
"~__8000
.time (min.) Fig. 2. First-order ph)t of the logarithm of remaining intensity (arbitrary units) versus time fi)r two amide protons that extensively overlap in chemical shift. The solid lines are the regression analysis [it for the exchange rate constant of the fast phase (Lys-32) and the slow phase (Trp-77). The intensity measurements were made in one-dimensional spectra, but the identification of the respective fast and slow phases was based upon cross peaks in two-dimensional spectra.
Fig. 3. The peaks have the same phase as cross peaks for scalar correlated protons, opposite to the phase of rotating frame NOE peaks, and lie at the chemical shift of the amide and the shift of water [19,20]. Sophisticated methods exist to discriminate these exchange cross peaks from true scalar correlation peaks [21], but in practice they are easy to identify if one records spectra at several different temperatures. The water resonance is usually much more temperaturesensitive than a proton resonances, and the exchange cross peaks always move in a band with the water peak.
.
9
G54=
0
0
K28o~
0
L74='
A65o~ $67~
0
D2X.D2=~
4.6 G39X
@
©)
A38X D68X~TA65X
D69X
o
4.8 G54X CI2¢x
!
T
9.4
!
9.3
4.4
D69¢~
9:2
T 9.1
"l 9.0
PPN
o'.9
81o
| 0.7
.:s
t 8.5
5.0
7 Pl~ 9.4
Fig. 3. A lx)rtion of the two-dimensional H O H A H A spectrum of P. aeruginosa c'-551. Cross peaks representing scalar coupling between the amide proton and the or proton are marked wtih the one letter code for the residue. Chemical exchange cross peaks with the water are marked with an X. They distinctively lie in a band at the water chemical shift in the FI dimension in this quadrant of data. The situation with Asp-2 illu.~trates how an a proton can overlap with the water re,~)nance.
13 to be observed, and, from our knowledge of assignments and NOESY spectra, there was no opportunity for an NOE involving an t~ proton under or near the water peak. Class VB represented amides that showed a marked decrease in intensity in saturation transfer experiments, but the magnitude is ambiguous because of the potential for an N O E involving an a proton. The rates reported assume that the observed decrease is solely due to saturation transfer. If there were a major NOE, for example say a 50% negative enhancement, then the effect would bc that the true exchange rate constant is 10-times smaller. Significant intensity changes were observed in saturation transfer experiments around 8.4, 7.9 and 7.5 ppm, but these could not be interpreted because of severe overlap and ambiguity involving NOE effects. There are class IVC protons in each of these ranges. Class VI represented the most slowly exchanging amides. A pH 7 sample sealed under argon had been kept for over 2 years at room temperature. It was not systematically studied for this time, but it was possible to measure the remaining intensity of the extremely slowly exchanging protons and perform a two point (times zero and 2 years) estimate of the rate. Class VIA protons had intensities of less than one, while VIB protons had intensities still within experimental error of one proton, and hence for practical purposes are non-exchangable. Class VII represented four protons for which not even approximate estimates could be made. This included the free amino terminus of Glu-I for which no assignment has been made. Phe-7, Tyr-27 and Ala-35 have been assigned, but they are in crowded regions in one-dimensional spectra. In H O H A H A spectra in ] H 2 0 ( n o exchange kinetics) they give weak cross -8
i
i
10
20
peaks because of unfavorable coupling constants between the amide and a protons. Based upon the crystal coordinates for c-551 the predicted constants are 1.7 (Phe-7), 3.0 (Tyr-27) and 6.2 Hz (Ala-35). The crystaI predicted 6.2 Hz value for Ala-35 is too high. A recent solution structure for P. aeruginosa c-551 based upon scalar and NOE constraints [24] suggests extensive differences between crystal and solution around residue 35 and our own COSY spectra indicate a small coupling constant, less than 4 Hz, for Ala-35. The H O H A H A cross peaks thus start out with an initial intensity that is low and were absent by the first H O H A H A spectra in ZH solvent. This is at first glanc~ like a class IVB proton, but the starting intensity was so weak that they could be exchanging slower and the residual cross peak intensity was obscured by noise. Side chain amide protons have been assigned in P. aeruginosa c-551. In general they are in crowded spectral regions where there are other rapidly exchanging protons. They arc observable in H O H A H A spectra in the aromatic region (see Fig. 3, Ref. 25) and are absent by the first kinetic two-dimensional data point. They could belong to class IVB or V, but there is insufficient data to so place them. They are not in any slow class. Discussion
The rates of exchange are presented in a graphical form in Fig. 4. Class VIIB protons for Fig. 4 were arbitrarily assigned a log k value of -7.5 to place them on the graph, but are specially marked. Class IVC protons were arbitrarily assigned a value of 2 for the same reason and are also specially marked. Other amides that have not been quantified or for which only an estimate has been made are omitted. However, bear
i
,
30
40
-6
$ -2
4.
i 0
residue
I
I
I
I
50
60
70
80
number
Fig. 4. Histogram representing the dislribulion of exchange rates in P. aen~ginosa c-551. Data have been takc~ .¢rom Table I. The open bars represent residues where a single valued measurement has been made. The solid bars represent the .slowest exchanging residues where only a limiting estimate of less than 10 -7 rain- 1 has been made. These were arbitrarily assigned a value of - 7 . 5 for display purpt~ses. The cross-hatched bars represent residues that show chemical exchange cross peaks in H O H A H A spectra. Although only a limit of greater than 10 z rain - ~ has been assigned to these nlles in Table I. they are depicted here with an arbitrary value of 2 for display purposes.
14 in mind that these arc amides that exchange too slowly for saturation transfer or two-dimensional chemical exchange effects, but are fast enuogh to be gone by the first spectra in "H-solvent. it is likely that their log k values are between - 1 and 1. A stereoview of c-551 is given in Fig. 5. Some of the present data can be compared to exchange rates determined for horse heart cytochrome c [6-9]. Cyt c is a larger protein, 104 residues, but it shares many features with c-551, especially the ligand geometry, and N- and C-terminal a helices. The N- and C-terminal residues exchange rapidly and this is perhaps to be expected. A segment from residues 35 to 43 shows consistently high exchange rates. The crystal structure of c-551 [I0] reported a type I turn from residue 34 to 37 and a type I1 turn from 37 to 40. Residues 41-43 mark the beginning of what was termed the 40's helix (41-50). In the crystal structure determination, this segment had some of the highest diffraction mean temperature factors in the entire protein (see especially Fig. 6 of Ref. 10). High temperature factors could represent either static disorder in the crystal or large mean amplitudes of thermal motion. The crystallographic data and the exchange data both mark this region as special in the structure of the protein. Residues 18-20 are in another fl bend on the protein surface and this could rationalize the relatively high exchange rates here. They also have above normal temperature factors in the crystal structure, but not as high as around residue 36. Residues 67-70 are at the very beginning of the C-terminal helix and are at least partially solvent exposed in the crystal structure. Once again Asp-68 has an above average temperature factor.
The slowest exchanging amides can be rationalized in broad terms by hydrogen bonding and solvent inaccessibility. The quantitative exchange data is consistent with the hydrogen bonding pattern for helix geometry (carbonyI of the nth residue to the amide of n + 4). However, the actual relative rates could not have been predicted. In the previous paragraph it was shown that very fast exchange rates correlated well with high mean temperature factors in the diffraction data. The correlation does not work for the very slow residues. They have a factor of approx. 10 A 2, which is about the average or median value. The heme ligands and residues around the thioether bonds are slowly exchanging. This has also been noted for cytochrome c. The Met-61 heine ligand appears distinctive in Fig. 4, but it is surrounded by proline residues at 58, 60, 62 and 63. Slow exchange pertains for ot helical segments, especially the helices from residues 27 through 34, the 40's helix from 40 through 51 and the C-terminal helix from 70 through 80. It was unexpected that the very slowest exchanging residues in c-551 were at the very ends of the 40's helix and C-terminal helix. There is nothing obvious in the crystal structure that earmarks these particular residues. Compare, for example, Leu44, a residue in the middle of the helix that orients its side chain toward the protein interior, with lle-48, which could be described in a similar way, and Lys-49, whose side chain extends into solution. The latter two residues have exchange rates 4-5 orders of magnitude slower than the former. It is possible that for unclear reasons, the ends of these two helices have especially stable hydrogen bonds. These slow residues have rates comparable to the slowest in horse cytochrome c and demonstrate that equivalent nonlabile amides can exist
Fig. 5. Stereoview of P. aeruginosa c-551 based upon the crystal coordinates of Re[. 10. The main chain atoms and carbonyl oxygen are shown to depict the folding paltern. The heine, heine ligands and thioelher bridges are shown to orient the active site. The N-terminal residue is at the top back and the N-terminal helix runs toward Ihe viewer. The 27-34 helix is visible on the lower right side and the 40's helix on the left side. The C-lerminal helix is at the back of Ihis view and runs diagonally a c r e s the view [rom the upper left to the lower right.
15
even in the smaller cytochrome. The end of the Cterminal helix is also the slowest segment in oxidized horse cytochrome c [9]. The 27-34 helix and the 40-50 helix in c-551 have no directly homologous elements in cytochrome c (ar~ informative comparison is Fig. 3 in Ref. 26) The 40's helix is loosely similar to a helix region in cytochrome c from 62-69, but the orientation and packing against the heme are different. Both cytochrome c and c-551 have an N-terminal a helix. In horse cytochrome c this is a region of very slow exchange 10 -~ to 10 -~ min-m for residues lie-9 and Phe-10 [6]. In c-551 it is not exceptionally slow, but moderate at around 10 -3 rain -~. The helix is inherently shorter in c-551 (a possible 12 residues till the first thioether versus 14 in cytochrome c) and Pro-3 shortens the possible length even further. This suggests greater flexibility in this region in the smaller protein. To summarize, we would like to stress the general conclusion that, in so far as protein stability is reflected by amide exchange rates, cytochrome c-551 is very comparable both overall and in specfics, to mitochondrial cytochrome c. Hypotheses that address the structure and function of c-type cytochromes should also address the fact that c-551 can achieve with circa 80 residues about the same thing as mitochondrial cytochrome c with over 100 residues. Acknowledgement This work was supported in part by grant C-~43292 from the National Institutes of Health. References 1 Tuchen, E. and Woodward, C.K. (1985) J. Mol. Biol. 185, 405-419. 2 Roder, H. and Wuthrich, K. (1986) Proteins !, 34-42.
3 Oiwen, W., Kline, A.D. and Wuthrich, K. (1987) Biochemistry 26. 6488-6493. 4 Udgaonkar, J.B. and Baldwin, R.L. (1988) Nature 335, 694-699. 5 Linse, S., Teleman, O. and Drakenberg, T. (1990) Biochemistry 29, 5925-5934. 6 Wand, A.J., Roder, H. and Englander, S.W. (1986) Biochemistry 25, 1107-1114. 7 Englander, J.j., Englander, S.W., Louic, G., Roder, H., Tran, T. and Wand, AJ. (1988)in Structure and Expression (Sarma, R.H. and Sarma, M.H., eds.), Vol. 1, pp. 107-117, Adenine Press. 8 Roder, H., EIove, G.A. and Englander, S.W. (1988) Nature 335, 700-704. 9 Jeng, M.F., Englander, S.W., EIoveo G.A., Wand, AJ. and Roder, H. (1990) Biochemistry 29, 10433-10437. 10 Matsuura, Y., Takano, T. and Dickerson, R.E. (1982) J. Mol. Biol. 156, 389-409. I I Timkovich, R., Dhesi, R., Martinkus, K.M., Robinson, M.K. and Rea, T. (1982) Arch. Biochem. Biophys. 215, 45-58. 12 Roder, H. (I989) Methods Enz~nol. 176A, 446-473K. 13 Chau, M.H., Cai. M. and Timkovich, R. (1990) Biochemistry 29, 5076-5087. 14 Detlefsen, DI., Thanabal, V., Pecoraro V.L. and Wagner, G. (1990) Biochemistry 29, 9377-9386. 15 Cai, M. and Timkovich, R. (1991) Biochem. Biophys. Res. Commun. 178, 309-314. 16 [Lax, A. and Davis, D.G. (1985) J. Magn. Re.c~n. 65, 355-360. 17 Pitner, T.P., Glickson, I.D., Dadok, J. and Marshall, G.R. (1974) Nature 250, 582-584. 18 Plateau, P. and Gueron, M. (1982) J. Am. Chem. Soc. 104, 7310-7311p. 19 Davis, D.G. and Bax, A. (1985) J. Magn. Reson. 64, 533-535. 20 Bay,, A., Sklenar, V., CIore, G.M. and Gronenborn, A. (1987) J. Am. Chem. Soc. 109, 6511-6513z. 21 Fejzo, J., Wcstler, W.M., Macura, S. and Markley, J. (1990) J. Am. Chem. Soc. 112, 2574-25771. 22 Sklenar, V. and Bax, A. (1987) J. Magn. Reson. 74, 469-479. 23 Perrin, C.L. and D ~ e r , T.]. (1990) Chem. Rev. 90, 935-967. 24 Detlefsen, D.J., Thanabal, V., Pecoraro, V.L. and Wagner, G. ( 1991 ) Biochemistry 30, 9{M0-9046. 25 Timkovich, R. (1990) Biochemistry 29, 7773-77801. 26 AImassy, R.J. and Dickerson, R.E. (1978) Proc. Nat. Acad. Sci. USA 75, 2674-2678.
