Fig. 6 - : Residues detected in the spectral region between amide I’ and amide II’ of deuterated RNase A dissolved in pure citric acid buffer: Image shows phase angles of components ( 1586cm , 1611cm , 1617cm and _1632cm for comparison) found near amide I’, image shows tyrosine components ( 1514cm , 1517cm ) near amide II’ comparing the different modulation frequencies/amplitudes of 9.3 mHz (dT/2=10°C), 37 mHz (dT/2=2.5°C) and 76.9 mHz (dT/2=1°C). The phase angles of the components are related to the phase angle of temperature. ab a b -1 -1 -1 -1 -1 -1 Fig. 3a-d: Amide I’ region of deuterated RNase A dissolved in pure citric acid buffer (0 M) and in buffer additionally containing 0.6 M, 1.2 M and finally 2.4 M urea: The image shows phase angles of five components ( beta 1632cm , random 1642cm , _random 1665cm , beta 1681cm , turn/random 1684cm ) for different modulation frequencies/amplitudes of 9.3 mHz (dT/2=10°C), 37 mHz (dT/2=2.5°C) and 76.9 mHz (dT/2=1°C). A phase angle of 0° corresponds to high temperature. a b c d -1 -1 -1 -1 -1 Fig. 8: Amide II’ region of deuterated RNase A dissolved in pure citric acid buffer: The image shows phase angles of six components ( 1373cm , 1407cm , _1424cm , 1442cm , 1459cm , _1474cm ) comparing the different modulation frequencies/amplitudes of 9.3 mHz (dT/2=10°C), 37 mHz (dT/2=2.5°C) and 76.9 mHz (dT/2=1°C). The phase angles of the components are related to the phase angle of temperature. -1 -1 -1 -1 -1 -1 Modulation at Frequency 9.3 mHz 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0.0 0.5 1.0 1.5 2.0 2.5 C13 urea concentration (mol/l) modulation amplitude Structural Changes upon Un-and Refolding RNase A as Monitored by FTIR ATR Temperature Modulation Spectroscopy I.Porth, D. Baurecht, U.P. Fringeli Institute of Physical Chemistry, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria Ribonuclease A (RNase A) is a model protein commonly used for folding studies. This single-domain enzyme (124 amino acids and 13.7 kD) has the advantage that it can be unfolded reversibly (thermally or by a chemical denaturant) to a certain extent. Native RNase A consists of about 26.6% alpha-helices (3 regions), 38% beta-sheet (6 strands) and additionally of turns and irregular protein segments. One method of dynamically observing structural changes during the refolding process of proteins is Fourier transform infrared (FTIR) spectroscopy. Time resolved FTIR studies have already been made [1]. The so-called peptide backbone and side chain infrared marker bands are known as sensitive monitors to conformational changes. The amide I' and the amide II' bands of RNase A show structural features, which origin from various overlapping components. The aim of this work was to resolve single components of the secondary structure by applying temperature modulation techniques to the reversible folding- denaturation process of RNase A. Ribonuclease A (RNase A) is a model protein commonly used for folding studies. This single-domain enzyme (124 amino acids and 13.7 kD) has the advantage that it can be unfolded reversibly (thermally or by a chemical denaturant) to a certain extent. Native RNase A consists of about 26.6% alpha-helices (3 regions), 38% beta-sheet (6 strands) and additionally of turns and irregular protein segments. One method of dynamically observing structural changes during the refolding process of proteins is Fourier transform infrared (FTIR) spectroscopy. Time resolved FTIR studies have already been made [1]. The so-called peptide backbone and side chain infrared marker bands are known as sensitive monitors to conformational changes. The amide I' and the amide II' bands of RNase A show structural features, which origin from various overlapping components. The aim of this work was to resolve single components of the secondary structure by applying temperature modulation techniques to the reversible folding- denaturation process of RNase A. REFERENCES [1] Reinstädler D., Fabian H., Backmann J., Naumann D. (1996) Biochemistry 35, 15822-15830 [2] Chirgadze Yu., Fedorov O., Trushina N. (1975) Biopolymers Vol. 14, 679-694 [3] Von Germar F., Dissertation (1998) [4] Fabian H., Mantsch H. (1995) Biochemistry 34, 13651-13655 [5] Susi et al. (1986) Biopolymers Vol. 25, 469-487 [6] Olinger J., Hill D., Jacobsen R., Brody R. (1986) Biochimica et Biophysica Acta 89-98 Introduction Materials and Methods FTIR ATR (attenuated total reflexion) technique was used to investigate structural changes of deuterated RNase A (20 mg/ml in 20 mM citrate/D2O, pD 4.2). Temperature modulations with different periods and amplitudes (108s/63°C 10°C, 27s/63°C 2.5°C, 13s/63°C 1°C) were performed at a mean temperature Tm=63°C (melting temperature at which half of the molecule is denatured) [1]. The temperature within the probe was determined by a significant baseline shift at 3100 cm calibrated by stationary spectra. To slow down the refolding process RNase A was dissolved in citrate buffer (20 mg/ml in 20 mM citrate/D2O, pD 4.2) containing 0.6 M, 1.2 M and 2.4 M C13-urea in further experiments. The spectroscopic response was analysed by digital phase sensitive detection (PSD). + + + -1 Fig. 2: Amide I’ region of deuterated RNase A dissolved in citric acid buffer: a) Stationary absorption spectrum at a temperature of 30°C (reference was buffer also meassured at 30°C); the adsorbed protein was subtracted to obtain only absorption by RNase A in solution; shown intensity of this spectrum is one tenth of the original. b yellow) Temperature modulation spectrum; modulation frequency being 9.3mHz and amplitude 10°C at a mean temperature of 63°C. c red) Resulting spectrum obtained by applying means of curve fitting to the original modulation spectrum. All other curves: Components obtained by curve fit procedure in this spectral region; this procedure was also carried out on modulation spectra resulting from different modulation frequencies/amplitudes and experiments with urea. + Table 1: Frequences of detected components and their half withs are listet. Components resolved during this work are put into comparison with data from literature on the subject. Fig. 7: Amide II’ region of deuterated RNase A dissolved in pure citric acid buffer: a) Stationary absorption spectrum at a temperature of 30°C (reference was buffer also meassured at 30°C); the adsorbed protein was subtracted to obtain only absorption by RNase A in solution; shown intensity of this spectrum is one tenth of the original. b yellow) Temperature modulation spectrum; modulation frequency being 9.3mHz and amplitude 10°C at a mean temperature of 63°C. c red) Resulting spectrum obtained by applying means of curve fitting on the original modulation spectrum. All other curves: Components obtained by curve fit procedure in this spectral region. + Fig. 5: Residues detected in the spectral region between amide I’ and amide II’ of deuterated RNase A dissolved in pure citric acid buffer: a) Stationary absorption spectrum at a temperature of 30°C (reference was buffer also meassured at 30°C); the adsorbed protein was subtracted to obtain only absorption by RNase A in solution; shown intensity of this spectrum is one tenth of the original. b yellow) Temperature modulation spectrum; modulation frequency being 9.3mHz and amplitude 10°C at a mean temperature of 63°C. c red) Resulting spectrum obtained by applying means of curve fitting on the original modulation spectrum. All other curves: Components obtained by curve fit procedure in this spectral region. + Results and Discussion In addition to the well known three components in the amide I' region (at about 1632 cm-1, 1667 cm-1and 1681 cm-1) obtained from temperature jump experiments, another one was found which appeared at higher urea concentrations with increasing modulation amplitude (at 1642 cm-1). At least three components of the amide II' region and some residues between the amide I’ and the amide II’ region could be detected. The tyrosine band observed at 1515 cm-1 in stationary absorption spectra splitted into two components upon temperature modulation confirming the shift of this band observed in earlier experiments [1]. The addition of the chemical denaturant with increasing concentrations to the enzyme resulted in a decrease of the modulation amplitude for all components except the one found at 1642 cm-1, whereas the phase angles of the detected components exhibited no significant change. Thus it seems that certain parts of the protein cannot join the refolding pathway but still there must exist some parts which are able to refold and they seem to do it at the same speed. The band at 1642 cm-1 is believed to be of random state and there are two possible explanations for its behavior: it is either unfolded from the beginning (random coil) or it hardly responds to the unfolding process. Interestingly enough this band seems to be in phase with the beta-sheet components. The phase angles decline with increasing modulation frequency and their calculation helps to find some correlations between certain components. As determined the phase angle of temperature is nearly the same as for random at 1664.6 cm-1 (and 1684.1 cm-1) while the two beta components (1632.3 and 1680.6 cm-1) are about 180° delayed. The tyrosine component at 1513.6 cm-1 is in phase with beta, the higher tyrosine band at 1517.3 cm-1 with random (temperature). Correlations between amide I’ and amide II’ components helped to assume amide II' band assignments. For instance there is some evidence for correlated behavior of the beta bands of amide I’ and bands of the amide II’ region at 1441.5 and 1473.5 cm-1. A component found at 1424.4 cm-1 could be the counterpart of amide I’ random coil. C13 Urea Concentration 0.0 M 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) C13 Urea Concentration 1.2 M 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) C13 Urea Concentration 2.4 M 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) wavenumber [cm -1 ] half with [cm -1 ] assignment reportedfrequencies [cm -1 ] &references 1684.1 33.8 turns, random 1684 [5] 1680.6 10.5 ß-sheet 1680 [4] ; 1681 [1] 1664.6 32.9 random 1665 [1] [5] ; 1662 [6] 1642.0 20.7 random 1646 [5] ; 1643 [6] 1632.3 19.9 ß-sheet 1633 [4] ; 1632 [1] [6] 1616.7 14.3 ? 1611.4 15.3 ? 1586.1 7.8 Arg(CN 3H 5 + v s); Asp(COO - v as) 1586 [2] ; 1584 [2] 1517.3 6.4 Tyr (ringC-C(v) +C-H(d)) 1515 [1] [2] 1513.6 15.5 Tyr (ringC-C(v) +C-H(d)) 1515 [1] [2] 1473.5 31.4 ß-sheet 1459.0 20.5 ? 1441.5 29.0 ß-sheet 1424.4 25.6 random 1407.4 11.0 Glu(COO - v s); Asp(COO - v s) 1407 [3] ; 1405 [3] 1373.1 11.5 ? Modulation at Frequency 37.0 mHz 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0.0 0.5 1.0 1.5 2.0 2.5 C13 urea concentration (mol/l) modulation amplitude Modulation at Frequency 76.9 mHz 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 2.5 C13 urea concentration (mol/l) modulation amplitude Fig. 4 - : Amide I’ region of deuterated RNase A dissolved in pure citric acid buffer (0 M) and in buffer additionally containing 0.6, 1.2 and finally 2.4 M urea: The images show modulation amplitudes ( - ) and phase angles ( - ) of five components ( beta 1632cm , random 1642cm , random 1665cm , beta 1681cm , _turn/random 1684cm ) comparing the four urea concentrations for different modulation frequencies/amplitudes of 9.3 mHz (dT/2=10°C), 37 mHz (dT/2=2.5°C) and 76.9 mHz (dT/2=1°C). A phase angle of 0° corresponds to high temperature. af ac df -1 -1 -1 -1 -1 Modulation at Frequency 9.3 mHz 0 50 100 150 200 250 300 350 400 0.0 0.5 1.0 1.5 2.0 2.5 C13 urea concentration (mol/l) phase angle (°) Modulation at Frequency 37.0 mHz 0 50 100 150 200 250 300 350 400 0.0 0.5 1.0 1.5 2.0 2.5 C13 urea concentration (mol/l) phase angle (°) Modulation at Frequency 76.9 mHz 0 50 100 150 200 250 300 350 400 0.0 0.5 1.0 1.5 2.0 2.5 C13 urea concentration (mol/l) phase angle (°) Residues within Amide I' Region 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) Components of Tyrosine 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) Components within Amide II' Region 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) C13 Urea Concentration 0.6 M 0 50 100 150 200 250 300 350 400 0 10 20 30 40 50 60 70 80 modulation frequency (mHz) phase angle (°) Fig. 1: Modulation spectra (0°-165°) of temperature modulated RNase A calculated by phase sensitive detection (PSD). Spectra show the amide I' and amide II' region originating from a temperature modulation experiment (f=9.3 mHz, dT= 10°C). The positions of resolved components of all experiments are indicated. + a a b a a a b, c d a b e c f b, c b, c c d b 165° 150° 135° 120° 105° 90° 75° 60° 45° 30° 15° 0° Acknowledgement We would like to thank Prof. Dieter Naumann and Dr. Diane Reinstädler (RKI Berlin) for support at the beginning of our experiments.