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Structural heterogeneity in proteins at cryogenic temperatures. Cooling rate dependence
Volume 216, number 3,4,5,6
CHEMICAL PHYSICS LETTERS
31 December 1993
Structural heterogeneity in proteins at cryogenic temperatures. Cooling rate dependence Kelvin Chu, G. Ulrich Nienhaus
’ and Robert Philipp
Department of Physics, University of Illinois at Urbana-Champaign, I1 10 West Green Street, Urbana, IL 61801, USA
Received 12 October 1993
The influence of the cooling rate on the structural heterogeneity of sperm whale myoglobin in solution at cryogenic temperatures was studied. Samples were either cooled slowly (0.03 K/s) or rapidly by immersing in liquid propane at 77 K, which yielded a cooling rate of more than 100 K/s. FlXR spectra of the stretch bands of the heme-bound CO showed that the population of the A substates depends on the cooling rate. The structural heterogeneity within each A substate was assessed by temperature-derivative spectroscopy (TDS), which yields the distribution of enthalpy barriers for CO rebinding after photodissociation. No significant changes of the barrier distribution were found. It is concluded that, within the range explored, the cooling rate plays a negligible role in the structural heterogeneity of protein solutions at cryogenic temperatures.
1. Introduction Many liquids can be cooled through the freezing point fast enough that crystallization can be prevented. The viscosity of the supercooled liquid increases strongly with decreasing temperature until it forms a glass below the glass transition temperature, T,.Glasses are arrested liquids, disordered systems that exhibit order only over nearest neighbor distances. Since the crystal is the state of lowest free energy, the supercooled melt is metastable. Proteins, on the other hand, are biological macromolecules that form crystals, in which they are ordered over macroscopic length scales. Therefore, the statement that glass-forming liquids and proteins share essential properties may not seem appropriate. However, owing to their design, protein molecules can assume a multitude of slightly different structures, called conformational substates (CS), so that the position of a particular atom in the unit cell of a protein crystal is not sharply defined, but may vary by up to a few 8ngstrSms [ 11. Consequently, disorder exists on the scale of the size of the molecule. It is this structural heterogeneity that gives rise to the glass-like properties of proteins. A single protein molecule may be ’ To whom correspondence should be addressed.
viewed as a small droplet of the supercooled liquid, or as a glass bead below T, Experimental evidence for the glassy nature of proteins has been obtained in many different ways by many different techniques: ( 1) The atomic mean-square displacements, (x2) in the X-ray structures are much larger for proteins than for simple organic molecules. A large residual disorder remains at low temperatures, indicative of structural heterogeneity [ 2,3]. (2) Absorption bands in the UV/VIS/IR regions are inhomogeneously broadened, as shown by spectral and kinetic hole-burning experiments [ 4-8 1. (3) Protein reactions at low temperatures are characterized by nonexponential time dependences as a result of barrier distributions. The functional heterogeneity has been explained by the existence of conformation substates [ 7,9]. (4) Transitions between conformational substates in proteins are nonexponential in time, and their temperature dependence often deviates from the Arrhenius law [ 7 1. (5) Metastability in proteins has been shown by pressure relaxation experiments [ 10,11 ] , (6) The temperature dependence of the low temperature specific heat has a linear term characteristic of two-level systems (TLS), which are frequently
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observed in amorphous materials [ 12 1. However, there is an important difference between liquids and proteins: In simple liquids, but not in proteins, an ordered phase with translational symmetry exists. This phase, which can be avoided by rapid cooling, is the true ground state of the system. The metastable, supercooled melt, on the other hand, has a rugged conformational energy landscape with many local minima [ 13 1. At high temperatures, the system will perform random walks through the landscape including regions at somewhat higher conformational energy. On slow cooling, the system descends into low-lying regions of the landscape and finally settles in a state with low energy (fig. 1) . On rapid cooling, the rate of cooling can exceed the rate of conformational transitions, and the system may become trapped in regions of high conformational energy. Therefore, the state of the system obtained by rapid cooling is not only metastable with respect to the crystalline phase, but also with respect to the state that is obtained by slow cooling. Since protein molecules lack translational symmetry, they do not possess a unique ground state but, like supercooled liquids, a rugged energy landscape with many CS. The particular CS in which a protein molecule will be trapped on cooling will depend on the cooling rate. Usually, Tg depends only weakly on the cooling rate, which is a consequence of the strong temperature dependence of the relaxation times. Therefore, cooling rates are often not explicitly stated in experimental reports. Barkalov et al. studied vitrification effects in lysozyme at various degrees of hyG /
- ,fost \ \ t
cooling
slow coolmg
;
cc
Fig. 1. Sketch of a one-dimensional cross section through the many-dimensional conformational energy landscape in proteins, as a function of a single conformational coordinate, cc. Upon slow cooling, the system can adiabatically relax into low energy states. At fast cooling rates, the system may get trapped in states of higher energy.
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dration as a function of cooling rate [ 141. From their data, the authors concluded that the cooling rate is an important parameter, and they asserted that “the existence of CS, of deviations from exponential time and temperature dependences of the relaxation rate and of a linear term in low-temperature specific heat can all appear as simple consequences of the abovementioned effects of vitrification”. We disagree with this statement, and since these issues are of crucial importance in our understanding of the structure and dynamics of proteins, we performed experiments to study the influence of the cooling rate on the structural heterogeneity of proteins which are reported in this Letter.
2. Experimental Studies were performed with sperm whale carbonmonoxymyoglobin (MbCO ) solutions. A potassium phosphate buffer solution (pH 6.7) and a mixture of 75% glycerol and 25% buffer (vol/vol) (pH 6.5) were used as solvents. The protein solutions were kept between two sapphire windows separated by a small mylar washer of 75 pm thickness. The sandwich was held in a tiny copper can of 4 mm height and 6 mm diameter. For slow cooling, the sample was attached to the cold finger of a closed cycle refrigerator and ramped down in temperature by a digital temperature controller with 0.03 K/s. For rapid cooling, the sample was immersed in liquid propane at 77 K and subsequently transferred to the refrigerator. In this way, a cooling rate of more than 100 K/s was achieved (see below). The structural heterogeneity of the sample was examined with Fourier transform infrared spectroscopy (FTIR) of the stretch bands of the heme-bound CO; their absorbances are proportional to the concentration of MbCO molecules. The distribution of rebinding barriers, which reflects structural differences in the protein molecules, was assessed with temperature derivative spectroscopy (TDS) [ 15,161. Details of the experimental setup are described in ref. [ 17 1.
3. Results and discussion The IR spectrum of the CO stretch absorption in
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sperm whale MbCO consists of three bands that correspond to three conformational substates, called A,,, Ai, and AS, which constitute the highest tier (CSO) in the hierarchy of conformational substates (fig. 2) [ 7,181. Within each of the A substates, a multitude of substates of tier 1 (CS 1) exists. The relative populations of the A substates depend on external parameters, such as temperature, pressure, pH, solvent composition and hydration degree [ 18-231. At sufficiently high temperatures, the A substates are in dynamic equilibrium. The temperature dependence of the fluctuation rates between A0 and Ai, A3 has been measured over almost 10 decades in rate [ 191. When cooling an MbCO sample in a glycerol/water mixture slowly (0.03 K/s) from room temperature, the population in A,, grows at the expense of the other two substates down to x 190 K. Below this temperature, the transitions become too slow compared to the cooling rate to establish equilibrium [ 201. When shock-freezing the sample with liquid propane, the sample is expected to fall out of equilibrium at a significantly higher temperature. Fig. 2 shows infrared difference spectra of the A substate lines at 12 K that were obtained with slow and fast cooling of the MbCO sample in 75% glycerol/25% buffer solvent. The shock-frozen sample contains less AO, The amount of A0 corresponds to that of an equilibrium spectrum taken at 220 K. Therefore, the glass transition temperature for the A substate interconversion is shifted up by x 30 K for the shock-frozen
0.05 ~
-0.2ot, 1900
I 1920
1940 Wovenumber
1 1960
I
I
1980
2000
(cd)
Fig. 2. mIR difference spectra of the stretch bands of the hemebound CO in sperm whale MbCO, showing the three A substates & (= 1966 cm-‘), AI (z 1945 cm-‘), and A3 (z 1930 cm-‘). The sample was cooled at 0.03 K/s (p) and x 100 K/s (---) from 260 to 77 K.
3 1 December 1993
sample. Before immersion in liquid propane, the sample was kept at 260 K. Since the fluctuation rate at 220 K is x0.3 s-l [ 191, we obtain a cooling rate of more than 100 K/s. The two spectra in fig. 2 give clear evidence for metastability in proteins, as has been shown previously with pressure-jump experiments [lO,ll]. While the results on the A substates were anticipated from the known transition rates, the more interesting question was how the heterogeneity within each A substate was affected by the cooling rate. We therefore examined the barrier distributions for ligand rebinding after photolysis with the TDS method. At 12 K, the samples were completely photodissociated with light from an Ar ion laser. The temperature was then ramped up linearly in time at a rate of 10 mK/s. FAIR spectra were taken continuously to monitor CO recombination. At low temperatures, only molecules with low enthalpy barriers recombine. As the temperature increases, rebinding becomes more and more probable for molecules with higher barriers. The negative temperature derivative of the population, -dN/d7’, approximates the enthalpy barrier distribution [ 15,161. Fig. 3 shows the results for the two MbCO samples studied. The data were normalized such that the sum of the areas below the three A substate curves equals one for the slowly cooled samples. The data of the flash-frozen samples were then normalized to have the same areas as the slowly cooled sample within each of the individual A substates, so that the shapes can be compared. Only very minor changes of the barrier distributions are apparent, although the cooling rates differ by almost than four orders of magnitude. The TDS data can be modeled with Gaussian distributions of enthalpy barriers within each A substate [ 151; results from nonlinear least-squares fits with Gaussians are summarized in table 1. The lit procedure uses the Arrhenius law to connect temperature and rebinding enthalpy. Data below 40 K were excluded since they may include significant contributions from quantum tunneling. The free energy barriers that separate CS in lower levels of the substate hierarchy are smaller, hence their fluctuation rates are faster, and the CSl maintain equilibrium down to x 160 K at cooling rates of 0.03 K, and down to = 190 K at cooling rates as high as 100 K/s. The mean-square displacements in the 277
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(0)
Y
0.020~’
”
0
20
1
I
40
’
80
60 Temp
100
120
(K)
Fig. 3. TDS data for the three A substates for MbCO dissolved in (a) 75% glycerol/2596 buffer (pH 6.5), (b) buffer (pH 6.7). The TDS signal, -dN/dT, approximates the enthalpy distribution for ligand rebinding. Cooling rate 0.03 K/s (p); o 100 K/s (- - -) . The areas under the dashed lines have been normalized to those under the solid lines to enable comparison of the shapes of the distributions.
X-ray structures of myoglobin do not change significantly between 160 and 190 K [ 21. These results are consistent with our findings: since the functional heterogeneity, i.e. the distribution of enthalpy barriers for CO binding has not changed, it is reasonable to assume that the structural heterogeneity has not changed either. By contrast, Barkalov et al. claim that low rates of freezing “increase the degree of crystallinity of the protein globule” [ 14 1. The obvious discrepancy between our results and those of Barkalov et al. may arise from the fact that the latter base their conclusions mainly on differential scanning calorimetry (DSC) experiments, which measure bulk properties of the sample. Therefore, they cannot distinguish between the protein and the solvent component. Properties of protein and solvent may be distinctly different, and processes associated with the solvent shell 278
3 1 December 1993
may erroneously be attributed to the proteins. Results from Rayleigh scattering of Mijssbauer radiation (RSMR) experiments were invoked to support the conclusions. Unfortunately, this method also has intrinsic problems distinguishing between the protein and the solvent component [ 24,251. The exothermic peak in fig. 2 of ref. [ 14 1, which was taken as evidence for an increase of “the degree of crystallinity of the protein globule” may well arise from crystallization of water that had no time to crystallize under fast freezing conditions. This scenario is supported by studies with experimental techniques that allow to separate between solvent and protein properties. NMR measurements on hydrated a-chymotrypsin and lysozyme samples [ 261 show the same general form of the temperature dependence of the longitudinal relaxation time T, for both proteins, with only minor differences in the details: r, is governed by the protein at temperatures below 180 K, where the water molecules are effectively frozen. Above 180 K, reorientation of water provides an additional mechanism for dipolar relaxation. Relaxation rates T I’ can be modeled assuming separate contributions from protein and solvent. The NMR experiments indicate that hydration water partially melts around 180 K, and it may subsequently recrystallize in a more stable ice phase. Doster and co-workers [ 27 ] performed DSC experiments with myoglobin and amino acid solutions and obtained experimental results similar to those reported by Barkalov et al. [ 141. To separate protein and solvent contributions, they used IR spectroscopy of the O-D stretch of deuterated samples. The IR measurements gave evidence that amorphous ice in myoglobin solutions melts in the temperature range between 180 and 260 K, in agreement with the NMR result. An exothermic peak seen in experiments with amino acid solutions that were cooled at different rates was interpreted as the freezing of water that did not have time to crystallize during fast cooling.
4. Conclusions The structural heterogeneity of proteins in solution is only weakly influenced when cooling the sam-
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CHEMICAL PHYSICS LETTERS
3 1 December 1993
Table 1 Parameters summarizing the rebinding enthalpy barriers for MbCO in 75% glycerol/25% buffer and aqueous buffer for two different cooling rates a) Solvent
glycerol buffer
aqueous
Cooling rate
Substate
slow
A0 Al A3
fast
log(‘4/s-‘)
T
Hpk
r
(g)
(kJ/mol)
(kJ/mol)
8.7 8.9 10.4
42,l 50.6 74.6
8.7 10.7 18.5
7.0 6.1 9.5
A0 Al A3
8.7 8.9 10.4
43.1 50.9 74.0
8.9 10.8 18.3
7.4 6.5 9.6
slow
Ao Al A3
8.7 8.9 10.4
39.7 47.3 65.2
8.1 10.0 16.2
8.3 7.5 9.7
fast
Ao A, A3
8.7 8.9 10.4
39.3 47.8 66.5
8.0 10.0 16.3
8.7 7.7 9.6
‘) Pre-exponentials, A, are taken from flash photolysis measurements [ 71. The temperatures Tpk correspond to the maxima of the TDS signals, - dN/dT. The Gaussian distributions of rebinding enthalpies are characterized by peak enthalpies Hpk with full widths at half maximum r. Errors on temperatures are f 0.5 K, and errors on enthalpies are k 0.2 kJ/mol.
ples with rates that differ by almost four orders of magnitude. The population of substates on the highest level of the hierarchy (CSO) depends on temperature. The glass transition temperature, T,, which marks the arrest of fluctuations within CSO, depends on the cooling rate; consequently, the population ratios of the A substates are affected by the cooling rate. On the lower tiers of the hierarchy, no significant changes could be inferred from the distributions of rebinding barriers. This result is consistent with the measurement of disorder by X-ray crystallography, which indicates that the mean-square displacements do not change much between 160 and 190 K, the glass transition temperatures within CS 1 for the two cooling rates.
Acknowledgement We thank all members of the Biological Physics Group for their collaboration. Special thanks are due to Professor H. Frauenfelder for continuous discussions and helpful comments on this manuscript. This work was supported in part by the National Science Foundation (Grant DMB87-16476)) the National Institutes of Health (Grant GM 1805 1), and the Of-
fice of Naval Research
(Grant
NO00 14-92-J- 194 1) .
References [I ] H. Frauenfelder, G.A. Petsko and D. Tsemoglou, Nature 280 (1979) 558. [2] F. Parak, H. Hartmann and G.U. Nienhaus, in: Protein structure: molecular and electronic reactivity, eds. R. Austin, E. Buhks, B. Chance, D. De Vault, P.L. Dutton, H. Frauenfelder and V.I. Goldanskii (Springer, Berlin, 1987) p. 65. [3] B.F. Rasmussen, A.M. Stock, D. Ringe and G.A. Petsko, Nature 357 (1992) 423. [4]S.G. Boxer, D.S. Gottfried, D.J. Lockhart and T.R. Middendorf, J. Chem. Phys. 86 ( 1987) 2439. [ 51 B.F. Campbell, M.R. Chance and J.M. Friedman, Science 238 (1987) 373. [6] P. Ormos, A. Ansari, D. Braunstein, B.R. Cowen, H. Frauenfelder, M.K. Hong, I.E.T. Iben, T.B. Sauke, P.J. Steinbach and R.D. Young, Biophys. J. 57 ( 1990) 19 1. [ 71 P.J. Steinbach, A. Ansari, J. Berendzen, D. Braunstein, K. Chu, B.R. Cowen, D. Ehrenstein, H. Frauenfelder, J.B. Johnson, D.C. Lamb, S. Luck, J.R. Mourant, G.U. Nienhaus, P. Ormos, R. Philipp, A. Xie and R.D. Young, Biochemistry 30 (1991) 3988. [ 8 ] J. Zollfrank, J. Friedrich and F. Parak, Biophys. J. 6 1 ( 1992) 716. [ 91 R.H. Austin, K.W. Beeson, L. Eisenstein, H. Frauenfelder and I.C. Gunsalus, Biochemistry 14 (1975) 5355.
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[ lo] I.E.T. Iben, D. Braunstein, W. Doster, H. Frauenfelder, M.K. Hong, J.B. Johnson, S. Luck, P. Ormos, A. Schulte, P.J. Steinbach, A. Xie and R.D. Young, Phys. Rev. Letters 62 (1989) 1916. 1” 1H. Frauenfelder, N.A. Alberding, A. Ansari, D. Braunstein, B.R. Cowen, M.K. Hong, I.E.T. Iben, J.B. Johnson, S. Luck, M.C. Marden, J.R. Mourant, P. Ormos, L. Reinisch, R. Scholl, A. Schulte, E. Shyamsunder, L.B. Sorenson, P.J. Steinbach, A. Xie, R.D. Young and KT. Yue, J. Phys. Chem. 94 (1990) 1024. 1’2 V.I. Goldanskii, Yu.F. Krupyanskii and V.N. Fleurov, Dokl. Akad. Nauk SSSR 272 (1983) 978. [13 F.H. Stillinger and T.A. Weber, Science 225 ( 1984) 983. 114 I.M. Barkalov, AI. Bolshakov, V.I. Goldanskii and Yu.F. Krupyanskii, Chem. Phys. Letters 208 (1993) 1. t15 1J. Berendzen and D. Braunstein, Proc. Natl. Acad. Sci. US 87 (1990) 1. [‘6 J.R. Mourant, D.P. Braunstein, K. Chu, H. Frauenfelder, G.U. Nienhaus, P. Ormos and R.D. Young, Biophys. J., in press. [ 171 G.U. Nienhaus, J.M. Mourant and H. Frauenfelder, Proc. Natl. Acad. Sci. US 89 (1992) 2902. [ 181 A. Ansari, J. Berendzen, D. Braunstein, B.R. Cowen, H. Frauenfelder, M.K. Hong, I.E.T. Iben, J.B. Johnson, P. Ormos, T.B. Sauke, R. Scholl, A. Schulte, P.J. Steinbach, J. Vittitow and R.D. Young, Biophys. Chem. 26 (1987) 337.
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[ 191 R.D. Young, H. Frauenfelder, J.B. Johnson, D.C. Lamb, G.U. Nienhaus, R. Philipp and R. Scholl, Chem. Phys. 158 (1991) 315. [ 201 M.K. Hong, D. Braunstein, B.R. Cowen, H. Frauenfelder, I.E.T. Iben, J.R. Mourant, P. Ormos, R. Scholl, A. Schulte, P.J. Steinbach, A. Xie and R.D. Young, Biophys. J. 58 (1990) 429. [21] M.W. Makinen, R.A. Houtchens and W.S. Caughey, Proc. Natl. Acad. Sci. US 76 (1979) 6042. [22] H. Shimada and W.S. Caughey, J. Biol. Chem. 257 (1982) 11893. [23] W.E. Brown, J.W. Sutcliffe and P.D. Pulsinelli, Biochemistry 22 (1983) 2914. [24] G.U. Nienhaus, H. Hartmann, F. Parak, J. Heinz1 and E. Huenges, Hyperfine Interactions 47 (1989) 299. [ 251 Yu.F. Krupyanskii, V.I. Goldanskii, G.U. Nienhaus and F. Parak, Hyperfme Interactions 53 (1990) 59. [26] E.R. Andrew, D.J. Bryant and T.Z. Rizvi, Chem. Phys. Letters 95 (1983) 463. [27] W. Doster, A. Bachleitner, R. Dunau, M. Hiebl and E. Ltischer, Biophys. J. 50 ( 1986) 2 13.
CHEMICAL PHYSICS LETTERS
31 December 1993
Structural heterogeneity in proteins at cryogenic temperatures. Cooling rate dependence Kelvin Chu, G. Ulrich Nienhaus
’ and Robert Philipp
Department of Physics, University of Illinois at Urbana-Champaign, I1 10 West Green Street, Urbana, IL 61801, USA
Received 12 October 1993
The influence of the cooling rate on the structural heterogeneity of sperm whale myoglobin in solution at cryogenic temperatures was studied. Samples were either cooled slowly (0.03 K/s) or rapidly by immersing in liquid propane at 77 K, which yielded a cooling rate of more than 100 K/s. FlXR spectra of the stretch bands of the heme-bound CO showed that the population of the A substates depends on the cooling rate. The structural heterogeneity within each A substate was assessed by temperature-derivative spectroscopy (TDS), which yields the distribution of enthalpy barriers for CO rebinding after photodissociation. No significant changes of the barrier distribution were found. It is concluded that, within the range explored, the cooling rate plays a negligible role in the structural heterogeneity of protein solutions at cryogenic temperatures.
1. Introduction Many liquids can be cooled through the freezing point fast enough that crystallization can be prevented. The viscosity of the supercooled liquid increases strongly with decreasing temperature until it forms a glass below the glass transition temperature, T,.Glasses are arrested liquids, disordered systems that exhibit order only over nearest neighbor distances. Since the crystal is the state of lowest free energy, the supercooled melt is metastable. Proteins, on the other hand, are biological macromolecules that form crystals, in which they are ordered over macroscopic length scales. Therefore, the statement that glass-forming liquids and proteins share essential properties may not seem appropriate. However, owing to their design, protein molecules can assume a multitude of slightly different structures, called conformational substates (CS), so that the position of a particular atom in the unit cell of a protein crystal is not sharply defined, but may vary by up to a few 8ngstrSms [ 11. Consequently, disorder exists on the scale of the size of the molecule. It is this structural heterogeneity that gives rise to the glass-like properties of proteins. A single protein molecule may be ’ To whom correspondence should be addressed.
viewed as a small droplet of the supercooled liquid, or as a glass bead below T, Experimental evidence for the glassy nature of proteins has been obtained in many different ways by many different techniques: ( 1) The atomic mean-square displacements, (x2) in the X-ray structures are much larger for proteins than for simple organic molecules. A large residual disorder remains at low temperatures, indicative of structural heterogeneity [ 2,3]. (2) Absorption bands in the UV/VIS/IR regions are inhomogeneously broadened, as shown by spectral and kinetic hole-burning experiments [ 4-8 1. (3) Protein reactions at low temperatures are characterized by nonexponential time dependences as a result of barrier distributions. The functional heterogeneity has been explained by the existence of conformation substates [ 7,9]. (4) Transitions between conformational substates in proteins are nonexponential in time, and their temperature dependence often deviates from the Arrhenius law [ 7 1. (5) Metastability in proteins has been shown by pressure relaxation experiments [ 10,11 ] , (6) The temperature dependence of the low temperature specific heat has a linear term characteristic of two-level systems (TLS), which are frequently
0009-2614/93/$ 06.00 0 1993 Elsevier Science Publishers B.V. All rights reserved. SSDZ OOOOS-2614(93)E1284-N
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observed in amorphous materials [ 12 1. However, there is an important difference between liquids and proteins: In simple liquids, but not in proteins, an ordered phase with translational symmetry exists. This phase, which can be avoided by rapid cooling, is the true ground state of the system. The metastable, supercooled melt, on the other hand, has a rugged conformational energy landscape with many local minima [ 13 1. At high temperatures, the system will perform random walks through the landscape including regions at somewhat higher conformational energy. On slow cooling, the system descends into low-lying regions of the landscape and finally settles in a state with low energy (fig. 1) . On rapid cooling, the rate of cooling can exceed the rate of conformational transitions, and the system may become trapped in regions of high conformational energy. Therefore, the state of the system obtained by rapid cooling is not only metastable with respect to the crystalline phase, but also with respect to the state that is obtained by slow cooling. Since protein molecules lack translational symmetry, they do not possess a unique ground state but, like supercooled liquids, a rugged energy landscape with many CS. The particular CS in which a protein molecule will be trapped on cooling will depend on the cooling rate. Usually, Tg depends only weakly on the cooling rate, which is a consequence of the strong temperature dependence of the relaxation times. Therefore, cooling rates are often not explicitly stated in experimental reports. Barkalov et al. studied vitrification effects in lysozyme at various degrees of hyG /
- ,fost \ \ t
cooling
slow coolmg
;
cc
Fig. 1. Sketch of a one-dimensional cross section through the many-dimensional conformational energy landscape in proteins, as a function of a single conformational coordinate, cc. Upon slow cooling, the system can adiabatically relax into low energy states. At fast cooling rates, the system may get trapped in states of higher energy.
276
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dration as a function of cooling rate [ 141. From their data, the authors concluded that the cooling rate is an important parameter, and they asserted that “the existence of CS, of deviations from exponential time and temperature dependences of the relaxation rate and of a linear term in low-temperature specific heat can all appear as simple consequences of the abovementioned effects of vitrification”. We disagree with this statement, and since these issues are of crucial importance in our understanding of the structure and dynamics of proteins, we performed experiments to study the influence of the cooling rate on the structural heterogeneity of proteins which are reported in this Letter.
2. Experimental Studies were performed with sperm whale carbonmonoxymyoglobin (MbCO ) solutions. A potassium phosphate buffer solution (pH 6.7) and a mixture of 75% glycerol and 25% buffer (vol/vol) (pH 6.5) were used as solvents. The protein solutions were kept between two sapphire windows separated by a small mylar washer of 75 pm thickness. The sandwich was held in a tiny copper can of 4 mm height and 6 mm diameter. For slow cooling, the sample was attached to the cold finger of a closed cycle refrigerator and ramped down in temperature by a digital temperature controller with 0.03 K/s. For rapid cooling, the sample was immersed in liquid propane at 77 K and subsequently transferred to the refrigerator. In this way, a cooling rate of more than 100 K/s was achieved (see below). The structural heterogeneity of the sample was examined with Fourier transform infrared spectroscopy (FTIR) of the stretch bands of the heme-bound CO; their absorbances are proportional to the concentration of MbCO molecules. The distribution of rebinding barriers, which reflects structural differences in the protein molecules, was assessed with temperature derivative spectroscopy (TDS) [ 15,161. Details of the experimental setup are described in ref. [ 17 1.
3. Results and discussion The IR spectrum of the CO stretch absorption in
Volume 216, number 3,4,5,6
CHEMICAL PHYSICS LETTERS
sperm whale MbCO consists of three bands that correspond to three conformational substates, called A,,, Ai, and AS, which constitute the highest tier (CSO) in the hierarchy of conformational substates (fig. 2) [ 7,181. Within each of the A substates, a multitude of substates of tier 1 (CS 1) exists. The relative populations of the A substates depend on external parameters, such as temperature, pressure, pH, solvent composition and hydration degree [ 18-231. At sufficiently high temperatures, the A substates are in dynamic equilibrium. The temperature dependence of the fluctuation rates between A0 and Ai, A3 has been measured over almost 10 decades in rate [ 191. When cooling an MbCO sample in a glycerol/water mixture slowly (0.03 K/s) from room temperature, the population in A,, grows at the expense of the other two substates down to x 190 K. Below this temperature, the transitions become too slow compared to the cooling rate to establish equilibrium [ 201. When shock-freezing the sample with liquid propane, the sample is expected to fall out of equilibrium at a significantly higher temperature. Fig. 2 shows infrared difference spectra of the A substate lines at 12 K that were obtained with slow and fast cooling of the MbCO sample in 75% glycerol/25% buffer solvent. The shock-frozen sample contains less AO, The amount of A0 corresponds to that of an equilibrium spectrum taken at 220 K. Therefore, the glass transition temperature for the A substate interconversion is shifted up by x 30 K for the shock-frozen
0.05 ~
-0.2ot, 1900
I 1920
1940 Wovenumber
1 1960
I
I
1980
2000
(cd)
Fig. 2. mIR difference spectra of the stretch bands of the hemebound CO in sperm whale MbCO, showing the three A substates & (= 1966 cm-‘), AI (z 1945 cm-‘), and A3 (z 1930 cm-‘). The sample was cooled at 0.03 K/s (p) and x 100 K/s (---) from 260 to 77 K.
3 1 December 1993
sample. Before immersion in liquid propane, the sample was kept at 260 K. Since the fluctuation rate at 220 K is x0.3 s-l [ 191, we obtain a cooling rate of more than 100 K/s. The two spectra in fig. 2 give clear evidence for metastability in proteins, as has been shown previously with pressure-jump experiments [lO,ll]. While the results on the A substates were anticipated from the known transition rates, the more interesting question was how the heterogeneity within each A substate was affected by the cooling rate. We therefore examined the barrier distributions for ligand rebinding after photolysis with the TDS method. At 12 K, the samples were completely photodissociated with light from an Ar ion laser. The temperature was then ramped up linearly in time at a rate of 10 mK/s. FAIR spectra were taken continuously to monitor CO recombination. At low temperatures, only molecules with low enthalpy barriers recombine. As the temperature increases, rebinding becomes more and more probable for molecules with higher barriers. The negative temperature derivative of the population, -dN/d7’, approximates the enthalpy barrier distribution [ 15,161. Fig. 3 shows the results for the two MbCO samples studied. The data were normalized such that the sum of the areas below the three A substate curves equals one for the slowly cooled samples. The data of the flash-frozen samples were then normalized to have the same areas as the slowly cooled sample within each of the individual A substates, so that the shapes can be compared. Only very minor changes of the barrier distributions are apparent, although the cooling rates differ by almost than four orders of magnitude. The TDS data can be modeled with Gaussian distributions of enthalpy barriers within each A substate [ 151; results from nonlinear least-squares fits with Gaussians are summarized in table 1. The lit procedure uses the Arrhenius law to connect temperature and rebinding enthalpy. Data below 40 K were excluded since they may include significant contributions from quantum tunneling. The free energy barriers that separate CS in lower levels of the substate hierarchy are smaller, hence their fluctuation rates are faster, and the CSl maintain equilibrium down to x 160 K at cooling rates of 0.03 K, and down to = 190 K at cooling rates as high as 100 K/s. The mean-square displacements in the 277
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(0)
Y
0.020~’
”
0
20
1
I
40
’
80
60 Temp
100
120
(K)
Fig. 3. TDS data for the three A substates for MbCO dissolved in (a) 75% glycerol/2596 buffer (pH 6.5), (b) buffer (pH 6.7). The TDS signal, -dN/dT, approximates the enthalpy distribution for ligand rebinding. Cooling rate 0.03 K/s (p); o 100 K/s (- - -) . The areas under the dashed lines have been normalized to those under the solid lines to enable comparison of the shapes of the distributions.
X-ray structures of myoglobin do not change significantly between 160 and 190 K [ 21. These results are consistent with our findings: since the functional heterogeneity, i.e. the distribution of enthalpy barriers for CO binding has not changed, it is reasonable to assume that the structural heterogeneity has not changed either. By contrast, Barkalov et al. claim that low rates of freezing “increase the degree of crystallinity of the protein globule” [ 14 1. The obvious discrepancy between our results and those of Barkalov et al. may arise from the fact that the latter base their conclusions mainly on differential scanning calorimetry (DSC) experiments, which measure bulk properties of the sample. Therefore, they cannot distinguish between the protein and the solvent component. Properties of protein and solvent may be distinctly different, and processes associated with the solvent shell 278
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may erroneously be attributed to the proteins. Results from Rayleigh scattering of Mijssbauer radiation (RSMR) experiments were invoked to support the conclusions. Unfortunately, this method also has intrinsic problems distinguishing between the protein and the solvent component [ 24,251. The exothermic peak in fig. 2 of ref. [ 14 1, which was taken as evidence for an increase of “the degree of crystallinity of the protein globule” may well arise from crystallization of water that had no time to crystallize under fast freezing conditions. This scenario is supported by studies with experimental techniques that allow to separate between solvent and protein properties. NMR measurements on hydrated a-chymotrypsin and lysozyme samples [ 261 show the same general form of the temperature dependence of the longitudinal relaxation time T, for both proteins, with only minor differences in the details: r, is governed by the protein at temperatures below 180 K, where the water molecules are effectively frozen. Above 180 K, reorientation of water provides an additional mechanism for dipolar relaxation. Relaxation rates T I’ can be modeled assuming separate contributions from protein and solvent. The NMR experiments indicate that hydration water partially melts around 180 K, and it may subsequently recrystallize in a more stable ice phase. Doster and co-workers [ 27 ] performed DSC experiments with myoglobin and amino acid solutions and obtained experimental results similar to those reported by Barkalov et al. [ 141. To separate protein and solvent contributions, they used IR spectroscopy of the O-D stretch of deuterated samples. The IR measurements gave evidence that amorphous ice in myoglobin solutions melts in the temperature range between 180 and 260 K, in agreement with the NMR result. An exothermic peak seen in experiments with amino acid solutions that were cooled at different rates was interpreted as the freezing of water that did not have time to crystallize during fast cooling.
4. Conclusions The structural heterogeneity of proteins in solution is only weakly influenced when cooling the sam-
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Table 1 Parameters summarizing the rebinding enthalpy barriers for MbCO in 75% glycerol/25% buffer and aqueous buffer for two different cooling rates a) Solvent
glycerol buffer
aqueous
Cooling rate
Substate
slow
A0 Al A3
fast
log(‘4/s-‘)
T
Hpk
r
(g)
(kJ/mol)
(kJ/mol)
8.7 8.9 10.4
42,l 50.6 74.6
8.7 10.7 18.5
7.0 6.1 9.5
A0 Al A3
8.7 8.9 10.4
43.1 50.9 74.0
8.9 10.8 18.3
7.4 6.5 9.6
slow
Ao Al A3
8.7 8.9 10.4
39.7 47.3 65.2
8.1 10.0 16.2
8.3 7.5 9.7
fast
Ao A, A3
8.7 8.9 10.4
39.3 47.8 66.5
8.0 10.0 16.3
8.7 7.7 9.6
‘) Pre-exponentials, A, are taken from flash photolysis measurements [ 71. The temperatures Tpk correspond to the maxima of the TDS signals, - dN/dT. The Gaussian distributions of rebinding enthalpies are characterized by peak enthalpies Hpk with full widths at half maximum r. Errors on temperatures are f 0.5 K, and errors on enthalpies are k 0.2 kJ/mol.
ples with rates that differ by almost four orders of magnitude. The population of substates on the highest level of the hierarchy (CSO) depends on temperature. The glass transition temperature, T,, which marks the arrest of fluctuations within CSO, depends on the cooling rate; consequently, the population ratios of the A substates are affected by the cooling rate. On the lower tiers of the hierarchy, no significant changes could be inferred from the distributions of rebinding barriers. This result is consistent with the measurement of disorder by X-ray crystallography, which indicates that the mean-square displacements do not change much between 160 and 190 K, the glass transition temperatures within CS 1 for the two cooling rates.
Acknowledgement We thank all members of the Biological Physics Group for their collaboration. Special thanks are due to Professor H. Frauenfelder for continuous discussions and helpful comments on this manuscript. This work was supported in part by the National Science Foundation (Grant DMB87-16476)) the National Institutes of Health (Grant GM 1805 1), and the Of-
fice of Naval Research
(Grant
NO00 14-92-J- 194 1) .
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