THE RLBL NEWSLETTER Regional Laser and Biotechnology Laboratories At the University of Pennsylvania D IRECTOR : P ROF . R OBIN M. H OCHSTRASSER Number 26 http://rlbl.chem.upenn.edu October 2002 Editorial A new grant period By Robin M. Hochstrasser I am very pleased to announce that the RLBL resource grant from NIH has been renewed as of August 2002. We look forward to more years of research activity with collaborators and users. Please let us know if there is a way we can enlarge the scope of your research. The principal core research activities in the future will involve the following: Infrared analogues of NMR through the amide-I modes; Coherent IR methods for structure fluctuations; IR pump-probe methods of vibrational dynamics; Transient IR probing of protein folding and conformational dynamics; Spectroscopy of single proteins and biological assemblies; and ultrafast measurements. In this and future issues of our Newsletter we will provide an overview of each one of these research continued on page 2, right column Feature Article Dynamics, Mode Coupling and Structural Constraints by Femtosecond Infrared Methods By Igor V. Rubtsov Department of Chemistry, University of Pennsylvania Introduction In recent experimental developments in two dimensional infrared spectroscopy of peptides and proteins 1-6 here at the RLBL we showed that there exist exciting new possibilities for such methods in the determination of structures and conformational dynamics of biologically interesting molecules. In these 2D IR methods, sequences of femtosecond infrared pulses are used to manipulate vibrational coherences in the amide-I band region of the IR spectrum, leading to knowledge of angular distributions of the amide groups and their coupling which relatate to their spatial arrangements. The same 2D IR experiment measures the dynamics of the distributions of amide-I vibrational frequencies, which are related to the distributions of structures that occur in equilibrium. 2D IR has great promise for biological applications 4,7,8 , so that it is vital to address a number of fundamental issues regarding the dynamical responses of molecular vibrations in solutions. For example: What are the main deactivation channels of vibrational excitation in proteins or peptides? How does the relaxation time of vibrational continued on page 2, left column I NSIDE T HIS I SSUE 1 Editorial 1 Feature Article: Dynamics, Mode Coupling and Struc- tural Constraints by Femtosecond Infrared Methods 8 Research Article: Motion of Single Molecules and Proteins in Tehalose Glass 13 RLBL Resources and Recent Publications 15 Application Form THE RLBL NEWSLETTER 1
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THE RLBL NEWSLETTER Regional Laser and Biotechnology Laboratories
At the University of Pennsylvania D I R E C T O R : P R O F . R O B I N M . H O C H S T R A S S E R
Number 26 http://rlbl.chem.upenn.edu October 2002
Editorial A new grant period
By Robin M. Hochstrasser
I am very pleased to announce that the RLBL resource grant from NIH has been renewed as of August 2002. We look forward to more years of research activity with collaborators and users. Please let us know if there is a way we can enlarge the scope of your research. The principal core research activities in the future will involve the following: Infrared analogues of NMR through the amide-I modes; Coherent IR methods for structure fluctuations; IR pump-probe methods of vibrational dynamics; Transient IR probing of protein folding and conformational dynamics; Spectroscopy of single proteins and biological assemblies; and ultrafast measurements.
In this and future issues of our Newsletter we will provide an overview of each one of these research
continued on page 2, left columnI N S I D E T H I S I S S U E
1 Editorial
1 Feature Article: Dynamics, Mode Coupling and Struc-
tural Constraints by Femtosecond Infrared Methods
8 Research Article: Motion of Single Molecules and
Proteins in Tehalose Glass
13 RLBL Resources and Recent Publications
15 Application Form
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Feature Article Dynamics, Mode Coupling and Structural Constraints by Femtosecond Infrared Methods
By Igor V. Rubtsov Department of Chemistry, University of Pennsylvania
Introduction In recent experimental developments in two dimensional infrared spectroscopy of peptides and proteins1-6 here at the RLBL we showed that there exist exciting new possibilities for such methods in the determination of structures and conformational dynamics of biologically interesting molecules. In these 2D IR methods, sequences of femtosecond infrared pulses are used to manipulate vibrational coherences in the amide-I band region of the IR spectrum, leading to knowledge of angular distributions of the amide groups and their coupling which relatate to their spatial arrangements. The same 2D IR experiment measures the dynamics of the distributions of amide-I vibrational frequencies, which are related to the distributions of structures that occur in equilibrium. 2D IR has great promise for biological applications 4,7,8, so that it is vital to address a number of fundamental issues regarding the dynamical responses of molecular vibrations in solutions. For example: What are the main deactivation channels of vibrational excitation in proteins or peptides? How does the relaxation time of vibrational
continued on page 2, right column
SLETTER 1
Feature article continued from page 1 Editorial continued from page 1
areas which we anticipate will have ever increasing utility in determining the structural content of dynamical processes in biology. However, the RLBL iready to respond to a wide range of laser related experiments and we look forward to hearin
s
g from you.
Mid IR pump-probe spectroscopy
By Thomas Troxler
The current issue contains two research related articles. In our feature article we report on new technological advances in femtosecond transient mid IR laser spectroscopy including an application to study the vibrational dynamics and mode coupling of model peptides. This report by Dr. Igor Rubtsov is a nice example that clearly shows how the development of new experimental techniques here at the RLBL drives new research and a deeper understanding of microscopic processes in biochemistry and biophysics.
The second research article by one of our staff members touches a new research area in the field of single molecule spectroscopy. Dr. Erwen Mei is reporting on results he got by looking at the motion of single proteins confined in the cavities of a trehalose glass. This is an important topic, since the immobilization of biomolecules is a primary step to successfully study these molecules by spectroscopic methods.
As always, you will also find a short summary of our current research activities as well as a list of recent publications following the research articles.
Visit our website We always would like to encourge you to visit our website at http://rlbl.chem.upenn.edu. An extended description of the resources is available to outside users, as is information about new instrumentation when it becomes available for collaborative or service research projects. Please don’t hesitate to contact us if you are interested in any of the instruments and approaches to discuss possible collaborative and service projects, or if you have any other question concerning our activities.
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excitations depend on the vibrational mode excited? What range of time scales is spanned by energy transfer between different modes of vibration in a protein? Should vibrations of the various C=O and C-N oscillators within a peptide be treated as localized, or delocalized? How much of the coupling of amide-I (mostly C=O stretch), amide-II (mostly C-N stretch) or amide-A (N-H stretch) modes is due to interactions between transition charges and how much to mechanical effects? In addition to the questions above, models for the potential energy surfaces of peptides suitable for generating mode coupling mechanism need to be more fully explored, which requires that experiments be developed to obtain coupling of peptide backbone modes. A significant improvement in the utility of 2D IR could be expected if the infrared pulse sequences were chosen to manipulate two or more different types of molecular vibration. This experiment could be done by constructing laser pulse sequences from infrared sources having different frequencies. Such “two color” experiments were recently proposed along with discussion of their response functions 9,10. Beside the amide-I mode, which is mainly the C=O stretch, the N-H stretch, or amide-A mode, is also a useful indicator of secondary structure 11,12. Therefore, a two-color 2D IR experiment involving the amide-I modes at 6 µm and the amide-A modes at 3 µm is expected to provide useful new information on peptide structure and dynamics. We have developed at the RLBL an infrared femtosecond transient absorption instrument. Two-color femtosecond infrared pump/probe spectroscopy has been used to study the vibrational mode coupling of amide-A and amide-I,-II. The present article is based on two papers 13,14 recently submitted to the Journal of Physical Chemistry. After an overview of theexperimental apparatus employed, we present some of the highlights of these first results of IR pump/probmeasurements of the coupling and angular relations of the amide-A, amide-I and amide-II modes in single amide units and between units connected by hydrogen bonds. This findings lead to a detailed microscopic description of structures and dynamics within these
e
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continued on the next page
Feature article continued
systems. These studies confirm the high potential for further applications of these experimental methods.
Materials and Methods
The mid-IR pump/probe transient absorption spectrometer was constructed at the RLBL. It is based on a Ti:sapphire laser system producing 130 fs pulses.Two IR optical parametric amplifiers and two separate difference frequency generators produce mid-IR beams tunable from 3 – 10 µm (at 6 µm: bandwidth 120 cm-1, pulse duration 150 fs). One is used as a pump that can be centered at the amide-A band (N-H or N-D stretching mode). The probe and reference pulses (bandwidth 160 cm-1, pulse duration 140 fs) were tunable across the range from 1300 to 3550 cm-1. The polarization of the these pulses was controlled by polarizers. Samples were held in a rotating CaF2 cell with pathlengths ranging from 100 to 500 µm.
Experimental Results and Discussion
The following paragraphs contain some of our first results using mid IR transient absorption spectroscopy as a new tool to investigate dynamics, mode coupling and structures in proteins. A basic understanding of a transient IR spectrum is therefore a necessary first step. Generally, when one vibration of the molecular system is excited to quantum number v = 1, the IR transient absorption spectrum is composed of transient absorption (v=1 → v=2), ground state(v=0→v=1) and stimulated emission peaks (v=1→v=0), where v indicates a vibrational quantum number of any vibration. The frequency of the v=1→v=2 transition is anharmonically shifted from the 0→1 transition and its extinction coefficient is twice as large as that of 0→1. This diagonal anharmonicity determines the shape of the diagonal transient spectrum, which will exhibit an absorption (i.e. positive ∆OD) on the low frequency side of the excited band and a bleach (i.e. negative ∆OD) on the high frequency side. In addition to the diagonal spectral features, transient signals in the regions of bands not directly excited may be observed. These are called cross-peak signals. The presence of these signals at the instant of excitation indicates that there is an off-diagonal anharmonicity ∆ij , caused by the coupling between mode i and mode j.
bleach
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A). Vibrational Energy Flow in Acetylproline-NH2
This part of the work is focused on determination of the energy relaxation pathways in the acetylproline-NH2 (AcProNH2) model dipeptide. Knowledge of the detailed vibrational energy relaxation pathways in peptides is essential for understanding biological mechanisms of vibrational energy transduction and chemical reactions in protein environments. The amide-I mode relaxes in ca. 1 ps in most cases, implying that it is strongly coupled to other internal modes. The mechanisms of vibrational energy transfermight be influenced by fluctuations in the energy gaps and couplings between the amide units. It is therefore necessary to determine experimentally not only the relaxation rates but also couplings between the peptide units and their distributions. The FTIR absorption spectrum of the model dipeptide AcProNH2 in a range from 1540 to 1725 cm-1 is shown in Figure 1. There are three prominent absorption bands in this spectral region, the amide-II, amine end band (A) at 1580 cm-1, and the two amide-I bands (B) and (C) at 1633 cm-1 (acetyl end) and at 1693 cm-1 (amine end), respectively. Each of the three bands was excited separately by a spectrally narrow, mid-IR excitation pulse (dotted lines in Figure 1), and the transient difference absorption (∆OD) spectra, cover-ing the region from 1520 to 1735 cm-1 was recorded
Figure 1: Mid IR spectrum of AcProNH2.
1540 1560 1580 1600 1620 1640 1660 1680 1700 1720
Wavelength / cm- 1
CB
A(amine end)Amide-II
Amide-I(acety l end)
(amine end)Amide-I
. H3C-C - N-CH-C - NH2
H2CCH2
CH2
O OFTIR
S
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Beside this main transient (diagonal) feature, signals at the A and C band locations (off-diagonal signals) are detected after selective excitation of the B band (see again in Figure 2). These off-diagonal peaks exist only because the A and C modes are interacting with the B mode. For two interacting modes, for example B and A, the off-diagonal spectrum in the frequency region of the A mode is determined by a pair of transi-tions: a bleach of the A mode at the ωA frequency and a transition from the B-excited state to a combination band B+A, which is anharmonically shifted due to
Figure 2 : Transient absorption spectra of three delay times of 0.4, 1.3, and 2.7 ps after excitation of the B band at 1633 cm-1. The FTIR spectrum is included as
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CBA
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OD
Wa venumber / cm-1
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interaction of these two modes, peaking at ωA-∆BA. Thevalue of the mixed-mode anharmonicity ∆BA can be determined from the amplitude and shape of the off-diagonal transient spectrum. Detailed analysis of the early time transient spectrum of AcProNH2 indicates that the off-diagonal anharmonicity is ca. 5.5 cm-1. It is important to determine anharmonicity from the spectra at early times, before significant vibrational relaxation of the originally excited state occurs, as it scrambles information. Interestingly at later times the A-band off diagonal feature broadens and shifts to lower frequencies, clearly evident in Figure 2. Exactly this kind of behav-ior is expected if there is a population transfer from state B to state A. This is a direct observation of the vibrational energy transfer. The slow decay of the transient spectrum at C band indicates that over time B population is transferred to other modes that are also strongly coupled to the C mode. The 10 ps decay time is assigned to vibrational cooling of the peptide, the time necessary to transfer excess of vibrational energy to the surrounding solvent molecules.
The Relaxation Dynamics in AcProNH2
The various time constants measured in the foregoing experiments could not be rationalized on the basis of only the three coupled levels A, B and C exchanging population with each other. The kinetic model required the introduction of other vibrational levels that could exchange energy and equilibrate with the three states being observed in the experiments: These are the reservoir states (R). Physically, they represent other modes of the system that are anharmonically coupled to the excited states A, B and C and become populated by relaxation to and from them. The global fit results are schematically summarized in Figure 3. The C to A energy transfer process having the time constant of 3.1 ps is a clear case where we locate an efficient relaxation pathway of the intramolecular vibrational energy redistribution process between the three amide levels. The A and C modes are the amide-I and amide-II modes of the same peptide that involve displacements of similar atoms. It appears likely that efficient amide-I/amide-II relaxation will be ageneral property of amides. In contrast, the B-to-A energy transfer channel, involving transfer betweetwo different sets of atoms, takes 10 ps, showing t
n hat
SLETTER 4
amide-I/amide-II relaxation may not be important if the modes are on adjacent units.
Anisotropy and Structural Constraints:
Transient anisotropy measurements allow one to obtain alignments of the transition moments of the pumped and probed modes. The pump/probe anisotropy is determined by the mean values of the squared cosines of the angles between the transition dipoles of the pumped and probed states: 2( ) 0.4 [cos ] ( )r t P R tθ= (1) where R(t) is an orientational correlation function and the average is over the distribution of internal angles. At delay times near zero, or in the range when the overall and the internal motion of the molecule can be neglected, the average of cos2 can be obtained directly from experimental anisotropy values, yielding direct structural information. In the case of AcProNH2, the polarization data was obtained for each of the three different excitation wavelengths. The anisotropy of the diagonal peaks were all close to 0.4 as expected from pumping and probing the same transition. However different values were observed for the cross peaks, ranging from –0.1 to 0.4. If the mole-cule frame transition dipole directions of the amide-I and -II transitions are assumed to be transferable from earlier work on other molecules, we can find structuresthat are consistent with these anisotropy measure-ments. The measured angles are consistent with a C7 internally H-bonded structure dominating the signals (see Figure 4). The dihedral angles which correspond
Figure 3 : Schematic representation of the major energy-transfer pathways in AcProNH2.
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to the experimentally determined structure (ϕ~-87° and ψ~65°), are close to the angles reportedAcProNHMe (-80°, 80°)
for 17.
B). Coupling of Amide-A and Amide-I/-II modes
In a second application of transient mid-IR spectroscopy at the RLBL, we are studying the vibrational coupling between amide-I/-II and amide-A modes with the use of the newly constructed mid-IR spectrometer. The amide-A mode, being the most localized mode in amide, can be used as a probe to obtain structural information on the H-bonding network as well as to characterize transition dipole directions of other modes in peptides. As no information is availableon the coupling strength of the amide-A and amide-I/II modes in any peptides of any structure, we invest-igated this coupling in simple dipeptides targeting different elementary structural units. The coupling of these modes in the same amide was also examined. Figure 5 shows the relevant parts of the FTIR spectrum of the three model compounds
Figure 4 : Experimentally determined angles of the C7 structure of AcProNH2 compared to calculated results (in brackets). See text for more details.
SLETTER 5
p
robe
AcAla(H)OMe, AcAla(D)OMe and AcProNHMe chosenfor these experiments. The spectra of the three differ-ent pump pulses used for excitation of the amide-A modes are also shown in Figure 5. Pump pulses with different spectra were used for selective excitation of the desired structure. For example, the amide-A regionof the AcProNHMe consists of two bands: one sharp
band at 3452 cm-1 caused by non-hydrogen bonded N-H groups and another broad and strong band at 3333 cm-1 caused by a C7 self-hydrogen-bonded structure15. Selective excitation of the hydrogen-bonded structure allows us to obtain angular and cou-pling data for this particular structure. An example of the two-color transient spectra for AcProNHMe is shown in Figure 6. Transient signals in the spectral region of all three modes, two amide-I and amide-II(amino end) modes, are observed, indicating interaction of these modes with the excited amide-A mode. For example, the transient signal at ~1670 cm-1
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b
aCO
NH O
OMe
. Me
Me b
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Amide-A
C=O st r.
AcAla(H)OMe
A
b
s
o
r b
a
n
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e
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NH
Me C
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ON
H2CCH2
. Me
H2C cAcProNHMe
Amide-I I
Pump freeH-bonded
Amide-I
Amide-A
Amino end
Acetyl end
Wavenumber / cm -1
b
a CO
ND O
OMe
. Me
Me
Pump
a
Amide-A
C=O s tr.
Amide-I
AcAla(D)OMe
*4
Figure 5 : Linear infrared absorption spectra of AcAla(D)OMe (a), AcAla(H)OMe (b), and AcProNHMe (c) in the selected region of the amide-I/-II and amide-A bands.
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(Figure 6) is due to a coupling of the amide-A and amide-I modes of the same amide (amino end). The transient signal at ~1625 cm-1 is due to a coupling of the amide-A(amino) and amide-I(acetyl) modes acrossthe hydrogen bond. Note that these signals corre-spond to the C7 self-hydrogen-bonded structure, whichis a model for the γ-turn motif (see also Figure 4). The N-H and C=O groups across the hydrogen bond are close to being perpendicular, which may account for the small coupling of the amide-A / amide-I modes involved in hydrogen bonding. The coupling strength of the amide-A and amide-II (amino end) modes is ~2.5 cm-1.
The linear spectrum of the AcAlaOMe dipeptide (see again Figure 5) in the amide-A region is very different from that of AcProNHMe. The amide-A band is not much shifted to lower frequencies; its small splitting may indicate the presence of more than one structure: non-hydrogen bonded and very weakly hydrogen bonded. The off-diagonal transient spectrum in the spectral region of the two amide-I modes (Figure 7) yields anharmonic couplings of the amide-A / amide-I modes on the same amide of 1.4 cm-1 and 1.6 cm-1 for H- and D-conformers, respectively, smaller than obtained for the AcProNHMe, where the N-H group is involved in the internal hydrogen bonding. The transient spectrum at ~1740 cm-1 is due to a coupling of the amide-A and carbonyl group on the ester end, which is expected to be sensitive to
Figure 6 : The diagonal and off-diagonal transient absorption spectrum of AcAla(H)OMe measured 300 fs after excitation into the amide-A band. The linear FTIR spectrum is included for comparison.
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-0.4
-0.2
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Am-II
28 cm-1 (19%)2.5 cm-1
∆ NH-CO:∆
NH=165 cm-1
5.5 cm-1
1.4 cm-1
3.5 cm-1
3.5 cm-1
Pumpfree NH
H-bonded
Am-I
N-H str.
FTIRAmino end
Acetyl end
∆ a
bs. /
mO
D
Wavenumber / cm-1
SLETTER 6
different structures of the peptide.
Using transient spectra with the probe beam polariza-tions parallel and perpendicular to the pump beam, we found two different C5 conformers: with the N-H group weakly hydrogen bonded to the carbonyl oxygen, C5(carbonyl), or to the ester oxygen, C5(ester), as shown in Figure 8. A larger off-diagonal anharmonicity was found for the C5(carbonyl) conformer, consistent with the absence of direct bonding between the N-H and C=O groups in the C5(ester) conformer. The alignment of the transition dipoles in the C5(carbonyl) also favors a larger through space electrostatic interaction for that case (see Figure 8). In summary, we have shown that two-color infrared pump-probe spectroscopy is an effective approach for obtaining structural information in peptides. The intermode anharmonic coupling constants for amide-A / amide-I modes have been found to be structure sensitive and lie in the range of a few cm-1. The vibrational relaxation induced intermode coupling has been revealed from observed time dependent spectral shifts. The vibrational lifetime of the amide-A mode is shortend upon forming an intramolecular hydrogen bond. The alignments of the transition dipole moments of amide-A and amide-I modes were found to depend on the molecular conformation and the occurrence of hydrogen bonding, consistent with previous experimental results and theoretical calculations. The data obtained for the coupling strengths and transition dipole alignments in different elementary structures
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0.0
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1.0
1.5 Amide-I
C=O str.
ν pump= 3455 cm-1
FTIR
AcAla(H)OMe
∆ a
bs. /
mO
D
Wavenumber / cm- 1
Figure 7 : The off-diagonal transient absorption spectrum in the amide-I region of AcAla(H)OMe 300 fs after excitation of the amide-A band.
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can now be used to study structures of larger peptides and proteins with very high time resolution, perhaps following a temperature jump. Work in this direction is in progress at the RLBL.
Literature
1. P. Hamm, M. Lim, W. F. DeGrado, and R. M. Hochstrasser, Proc. Natl.. Acad. Sci. USA 96, 2036 (1999).
2. M. T. Zanni, S. Gnanakaran, J. Stenger, and R. M. Hochstrasser, J. Phys. Chem. B 105, 6520 (2001).
3. M. T. Zanni, N.-H. Ge, Y. S. Kim, and R. M. Hochstrasser, Proc. Natl. Acad. Sci. USA 98, 11265 (2001).
4. N.-H. Ge, M. T. Zanni, and R. M. Hochstrasser, J. Phys. Chem. A 106, 962 (2002).
5. S. Gnanakaran,R. Hochstrasser, Biophys. J. 80, 1254 (2001).
6. P. Hamm and R. M. Hochstrasser, In Ultrafast Infrared and Raman Spectroscopy; M. D. Fayer (ed.); NY, p. 273 (2000).
7. R. M. Hochstrasser, Chem. Phys. 266, 273 (2001).8. M. T. Zanni, S. Gnanakaran, J. Stenger, and R. M.
Hochstrasser, J. Phys. Chem. B 105, 6520 (2001).9. S. Mukamel, Principles of nonlinear spectroscopy,
Oxford Univ. Press, NY (1995). 10. S. Woutersen, H. J. Bakker, Science 278, 658
(1997). 11. K. Tonan and S. Ikawa, J. Am. Chem. Soc. 118,
6960 (1996). 12. D. T. McQuade, S. L. McKay, D. L. Powell, and S.
H. Gellman, J. Am. Chem. Soc. 119, 8528 (1997). 13. I. V. Rubtsov and R. M. Hochstrasser, J. Phys.
Chem. B 106, 9165 (2002). 14. I. V. Rubtsov and R. M. Hochstrasser, J. Phys.
Chem. B (submitted, 2002). 15. J. Neel, Pure Appl. Chem. 31, 201 (1972). 16. T. Miyazawa, J. Mol. Spectrosc. 4, 168 (1960). 17. V. Madison and K. D. Kopple, J. Am. Chem. Soc.
102, 4855 (1980).
Figure 8 : Skeletal diagram of AcAlaOMe in two conformations: C5(carbonyl) and C5(ester). Arrows indicate the measured directions of the amide-A, amide-I, and ester carbonyl transition dipoles.
C5 (ester) C5 (carbonyl)
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R E S E A R C H A R T I C L E :
M O T I O N O F S I N G L E M O L E C U L E S
A N D P R O T E I N S I N T R E H A L O S E
G L A S S
By Erwen Mei, Jianyong Tang, Jane M. Vanderkooi, and Robin M. Hochstrasser Chemistry and Biophysics Departments, University of Pennsylvania
We have carried out some single molecule experi-ments on free porphyrin cytochrome-c (P Cyt c) and Zn porphyrin cytochrome-c (Zn Cyt c) molecules en-capsulated in trehalose glass using single-molecule with a confocal microscope that records simultane-ously fluorescence signals in two orthogonal polariza-tion directions. Large angular motions were observed on time scales ranging to many seconds. The tre-halose glass restricts the accessibilty of oxygen to the fluorophore. An instrument of this type is now availablefor users and collaborative projects at RLBL. A summary of the single molecule resources at RLBL is given on page 12 of this Newsletter.
Introduction Water is essential for life: dehydration generally inactivates proteins and enzymes. However, pollen, seeds, fungal spores, and a variety of microscopic animals can survive dehydration for decades and restore activity within minutes of rehydration1. The secret of the survival from anhydrobiosis of many organisms is presence of the disaccharide α,α-trehalose (α−D-glucopyranosyl-α−D-glucopyranoside, see Figure 1). The mechanism by which trehalose exerts protection has been widely explored 2. Spectro-scopic studies indicated that trehalose replaces water molecules in forming hydrogen bonds to the surface of the protein 3,4. Trehalose also helps to prevent the loss of internal water molecules of protein 2. It appears that the glass forming ability of trehalose is essential for its bioprotective properties 5-7.
Trehalose forms viscous solutions and glasses at room temperature that are useful in studies of protein dynamics such as the kinetics of geminate recombination of carbon monoxide to myoglobin 2. In another example, when bovine rhodopsin was
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suspended in trehalose-water glass films, it was possible to trap intermediates in the light-activation process allowing for facile room temperature investigations of the spectroscopic properties of rhodopsin’s photointermediates 8. These examples show that proteins can function in trehalose glass.
Recent developments in single molecule spectroscopy provide effective methods for studying structural and dynamical features of individual molecules interacting with their surroundings 9-11. Such measurements yield direct information on the distributions of properties that exist in the equilibrium ensembles. We report here on the fluorescence polarization properties of single proteins in trehalose glass. The results allow the characterization of the distribution of environments or cavities in which the proteins are confined and the range of physical properties exhibited by the proteins. A large number of single molecule measurements can also be averaged to yield something approaching a bulk response whose underlying microscopic dynamics at equilibrium is known.
Summary of Experimental Methods The scanning confocal microscope, available in RLBL, uses a sample-scanning stage with closed-loop X,Y feedback for accurate sample positioning and location of individual molecules 12. The stage is mounted on an inverted, epi-illlumination microscope with a 1.3 numerical aperture objective. Circularly polarized 514.5nm and 488nm lines of an argon ion laser were used in separate experiments to excite the molecules. The excitation power in each case was in the range of 0.3µW-1µW.
Two avalanche diodes detected the photons reflected
Figure 1 : Structure of α,α-trehalose.
S
LETTER 8
and transmitted by a polarizing beam splitter, thereby isolating the fluorescence emitted by the molecules into two orthogonal polarization directions, s and p, in the plane of the microscope. The p-polarized fluorescence image, obtained by raster scanning the sample pixel by pixel, was used to locate molecules. Once located, a single molecule fluorescence spot was centered in the laser focus, and signals were collected in the two orthogonal polarization directions, binned into 1 ms intervals. The fluorescence spectra ofsingle molecules were obtained by means of a monochromator equipped with a back-illumination liquid-nitrogen-cooled CCD camera.
Thin films were prepared by spin casting 20µl of the protein-trehalose solutions onto a microscope cover slide, and drying in air. The water content in the trehalose film was measured from the infrared spectra of the films, which showed the molar ratio of water to trehalose to be 2 + 0.2. This is comparable with the water of hydration in crystalline.
Discussion of Results Figure 2 above shows a typical computer screen image of single metal free porphyrin cytochrome c in a trehalose glass. Each fluorescence point represents the collected signal from an individual molecule.
Figure 2 : Fluorescence image of single metal free porphyrin cytochrome c molecules in a trehalose glass.
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Individual time-resolved signals of both polarizations, so called intensity-time records, for many single molecules were recorded and stored on the computer. Figure 3 shows two examples of polarization resolved intensity-time traces from metal-free porphyrin cytochrome c (P-Cyt-c) in trehalose glass. Similar traces were also recorded from rhodamine 6G (R6G) and Zn-porphyrin cytochrome c (Zn-Cyt c) but are not shown here. Remarkable differences in these traces could be found, highlighted in Figure 3. The upper pair of traces, which correspond to the two polarization directions s and p, reveals some motion of the single molecule on long time scales, representative by a switching in the intensity pattern of the two polarizationdirections. In contrast, the lower pair of curves shows a relatively fixed molecule, whose mean polarization ratio is not changing much over the time during which emission was observed.
The chromophors of P-Cyt c and R6G are known to belinear oscillators for which the absorption and fluorescence step involve a single transition dipole. On the other hand, bulk samples of Zn-Cyt c in trehalose reveal a fluorescence anisotropy of 0.1±0.01, indicating a circular oscillator on average. If the emitting transition dipoles are fixed in, all records of intensity-time traces should exhibit a significant p-
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020151050
Figure 3 : Two different traces of the polarization resolved fluorescence intensity-time records of single P-Cyt c in trehalose glass.
SLETTER 9
signal for all times. Indeed, a certain percentage of recorded traces showed this behavior: 6.4% out of a total of 171 R6G molecules observed, 2% out of 159 folded and 14% out of 222 unfolded Zn-Cyt c molecules. Otherwise the intensity-time records showed the more prevalent alternation between the two orthogonal polarization directions as seen in the upper part of Figure 3.
Further indication of the chromophore’s movement within the trehalose cavity was found by turning the laser light quickly on and off. When the laser light was turned off for a short period of time, the polarization was generally different from when it was turned back on again. But the intensity-time records for different single molecules showed very little systematic behavior that was repeated from record to record. Some common features seen were slow (> 5s) signal variations during which the polarization alternates around a relatively well defined mean value. Or molecules were seen to enter periods during which there is no detectable emission: we refer to these as dark states.
We also investigated photo-bleaching properties of thevarious chromophores deposited either directly on a glass surface or confined in the trehalose glass. Photo-bleaching can be seen in Figure 3 as a permanent loss of fluorescence signal. We found that
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Figure 4 : Historgrams of the survival time for a set of rhodamine 6G molecules on a glass surface (upper frame) or in trehalose glass (lower frame).
THE RLBL NEW
R6G molecules in trehalose emit on average almost 4 times more photons before photobleaching than do their counterparts on glass. Fluorescence intensity-time records could also be used to calculate the total time prior to permanent photo-bleaching under continuous illumination using a fixed source intensity. The measured survival times of P-Cyt c and Zn-Cyt c uner the same illumination conditions were extracted, and it was found that many more P-Cyt c molecules live for times exceeding 20 s.
The total photons emitted by single molecules give important information about the nature and formation of the trehalose glass. The results in Figure 4 show that the total number of photons emitted (on average) by R6G single molecules is significantly greater in trehalose than on a glass surface. Also the average survival time of the molecules in trehalose film is about 3 to 4-fold larger than on a surface. The increase in total number of photons emitted results from the increased photostability. The increased dye stability observed in the trehalose film is caused by a reduction in the oxygen permeability and mobility.
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Figure 5 : Histogram plot of the fluctuations of dichroism in the intensity-time records observed from P-Cyt c (a) and
S
Zn-Cyt c (b) and (c). See text for further details.
LETTER 10
THE RLBL NEWSLETTER 11
In order to better understand the parameter distributions necessary to describe single molecule and single protein behavior in the glass cavities, we investigated the dichroism of the fluorescence signal A(t). The dichroism itself is defined as the ratio of the sum and difference of the p and s components of the fluorescence signals. Because of the expected heterogeneity we did not attempt to find a unique description that encompasses all the single molecule behavior of the dichroism. The histograms in Figure 5 and 6 document the extent of this heterogeneity and assist in the investigation of the statistical significance of the differences in the observed patterns for the three molecules studied. It is noteworthy that contributions from molecular motion are manifested in the fluctuations of the intensity-time records, but that these fluctuations also contain a significant amount of Poisson noise that tends to obscure contributions from molecular motion.
The histograms in Figure 5 of the fluctuation in the dichroism for each molecule defined by
22( ) ( )A A t A tδ = −
provide a very qualitative view of the dichroism variations. The variations also depend on the length of the intensity-time signal recorded (Figure 5c).
In the case of a linear oscillator the dichroism is
( ) cos(2 )A t φ=
where φ is the azimuth angle in the orthogonal laboratory coordinate system (s, p, N). The plot of N(A) should therefore show peaks in the distribution1 and –1. For a circular oscillator the distribution of Avalues should peak at 1, 0 and –1 with probabilities of 1:2:1. However the dynamic range of our experiment was such that the largest measurable intensity ratio was around 5. This implies that peaks in the distribution of A should be at ±0.63 instead of ±1.
at
Figure 6 shows these distributions of A computed over the first 5 s of each sample’s record with a binning length of 50 ms. If the molecules were fixed the distribution would peak between A = 0 and A = 1 because the choice of the molecule in the first place was determined by its visibility in p-polarization. Other molecules also show peaks between A = 0 and A = -1 indicative of motion. When A reaches a value near to
zero the shot noise causes the s and p signals to alternate in intensity as if there were fluctuations in A about zero that have the appearance of very fast (10’s of ms) restricted rotational motions.
el
nd
s well as
s been
olecules in
oles of
time scales ranging into the many seconds regime
research was supported by NIH grants PO1 to
Conclusions
In summary, we have built at RLBL a two channpolarization confocal microscope and used it to investigate the reorientations of single molecules aproteins in trehalose glass. Information about the formation and the nature of trehalose glass athe interactions between the dopant and its surrounding trehalose microenvironments haobtained by analyzing the photostability and orientational motions of proteins and dye mtrehalose. The entrapped molecules are approximately 3.4 times more stable than their counterparts on glass surfaces which evidences thelow oxygen permeability/mobility in trehalose glass. Significant angular motions of the transition dipsingle Rh6G, P Cyt-c and Zn Cyt-c molecules encapsulated in trehalose have been observed on
Acknowledgment This
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1 00
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b
c
AN(A)
Figure 6 : Experimental distributions of A(t) computed over the first five seconds for P-Cyt c (a), Zn-Cyt c (b), and R6G (c). See text for further details.
JV.
Literature
1. F. H. Crowe and L. Crowe, Ann. Rev. Physiol. 579, (1992).
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65, 661 (1993). ,
Sanchez, l. 47, 1391 (1996).
96). 10.
12. D. Howard, R. J. Cogdell, and R. M. Hochstrasser, Proc. Natl. Acad. Sci. USA 96, 11271 (1999).
C U R R E N T I N G L E - M O L E C U L E
2. G. M. Sastry and N. Agmon, Biochemistry 36, 7097 (1997).
3. J. F. Carpenter and L. Crowe, Biochemistry 28, 3916 (1989).
4. S. J. Prestrelski, N. Tedeschi, T. Arakawa, and J. F. Carpenter, Biophys. J.
5. J. L. Green and C. A. Angell, J. Phys. Chem. 932880 (1989).
6. D. P. Miller, J. J. D. Pablo, and H. R. Corti, J. Phys. Chem. 103, 10243 (1999).
7. S. P. Ding, J. Fan, J. L. Green, Q. Lu, E. and C. A. Angell, J. Therm. Ana
8. S. Sikora, A. S. Little, and T. G. Dewey, Biochemistry, 33, 4454 (1994).
9. X. S. Xie, Acc. Chem. Research 29, 598 (19S. Weiss, Science 283, 1676 (1999).
11. E. Mei, A. M. Bardo, M. M. Collinson, D. A. Higgins, J. Phys. Chem. B 104, 9973 (2000). M. A. Bopp, A. Sytnik, T.
S
T H E R L B L
After having presented a research article about an on-going single-molecule spectroscopy project, we would like to take
the RLBL.
We currently have two multi-channel, confocalmicroscopes using sample-scanning stages. Also available are setups for a near-field scanning microscope as well as an atomic force microscope. Various detectors can be fitted on both multi-channel microscopes. We have several avalanche photodiod(ADP) detectors, which are used for single-molecule spectroscopy experiments in the wavelength range from 500 – 900 nm. Incorporation of polarizing optics and optical filters allows for polarization and sp
S P E C T R O S C O P Y R E S O U R C E S A T
the opportunity and summarize resources available for single molecule spectroscopy research at
es
ectral
THE RLBL NEW
separation of the fluorescence signal before detectioUsing a pulsed excitation source and the time-correlated single–photon detection method, single- molecule fluorescence lifetimes can also be recorded.Recent upgrades for both instruments furthermore include the addition of lifetime imaging and mements of photon lifetime trajectories. These additions
n.
asure-
upled device (CCD) detector also allows for
blue diode laser
nts. We
n,
le
cules can be measured using a two-
o-
-
imaging is another method to distinguish several molecular com
greatly enhance our capabilities to determine experimentally single molecule lifetime distributions.
The incorporation of a monochromator into the detection pathway and use of a liquid nitrogen cooled charge-cothe recording of single molecule fluorescence emission spectra.
All microscope setups are accessible by various excitation sources present in our laboratory. We currently have cw Argon-ion (457, 488, and 514 nm) and HeNe (633 nm) lasers, various cw-diode lasers (800 – 850 nm), a pulsed picosecond Nd-YAG laser (355 and 532 nm), synchronously pumped dye laser(545 – 900 nm), a pulsed picosecond(408 nm), and a femtosecond Ti:sapphire oscillator (770-900 and 380-450 nm) present.
Combining these different excitation and detection capabilities allows us to perform several standard fluorescence based single molecule experimewould like to mention especially fluorescence resonantenergy transfer (FRET) studies to determine intramolecular distances between donor and acceptor molecules, measurements of single molecule fluorescence decay times and its distribution functioand fluorescence correlation spectroscopy to measure diffusion processes and chemical kinetics of singmolecules in solution. Time-resolved anisotropies ofsingle molechannel setup with separated polarization detectionpathways.
Furthermore, both instruments are also capable of performing single cell imaging of stained cells or cellaggregates. Again, spectral resolution as well as twphoton induced fluorescence experiments allow for studies of binding sites and molecular distributionswithin a single cell and the observation of the timeresolved evolution of chemical gradients. Lifetime
ponents present within a single cell.
SLETTER 12
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C U R R E N T T E C H N O L O G I C A L
D E V E L O P M E N T A N D R E S E A R C H
A T T H E R L B L
The main subjects under investigation at RLBL are shown below. If your research may be interfaced with any of these approaches we urge you to contact us. A full description of each of these topics is also available at our Web site http://rlbl.chem.upenn.edu.
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Dynamics of photoactivatable proteins and other biological structures: Methods are being developed to examine the responses of biological systems to light by pump/probe and nonlinear spectroscopic methods encompassing spectral regimes from the UV to the far IR and covering femtosecond to second timescales. Techniques include: single and multiple wavelength transient spectroscopy (UV/Vis, vibrational IR, Terahertz), photon echoes, two photon absorption and time-correlated single photon counting.
Methodologies to investigate protein folding and macromolecular conformational dynamics: Detection and characterization of intermediate states in conformational dynamics and unfolding isanother developing technology at RLBL. Laser-based temperature-jump instruments are available for these investigations.
Investigations of single molecular assemblies using confocal and atomic force microscopes: It is now possible to examine the properties of single molecules using fluorescence in association with confocal microscopy. The RLBL is coupling single molecule detection methods with mature time correlated photon counting technology, polarization scanning and pulsed laser experiments.
Energy transfer and fluorescence monitoring of biological dynamics: Monitoring fluorescence lifetimes and anisotropies reveals details of proteindynamics. Techniques are being developed at the RLBL to monitor these properties of fluorescing species on the femtosecond to nanosecond timescale.
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and
Development of time resolved far-IR (terahertz) probes for protein dynamical changes: New powerful sources of THz and far-IR radiation are developed and used as laboratory THz source.
Two-dimensional infrared spectroscopy and infrared analogues of NMR: Heterodyned photon echo spectroscopy and spectrally resolved three pulse IR photon echoes are employed to investigate the amide I region and other transitionsof peptides and small proteins.
S E L E C T I O N O F R E C E N T
B L I C A T I O N S
ational dynamics, mode coupling and structural straints for acetylproline-NH2. I. V. Rubtsov and R. ochstrasser, J. Chem. Phys. B 106, 9165 (2002).
rescence quenching and lifetime distributions of le molecules on glass surfaces. M. Lee, J. Kim, J. g, and R. M. Hochstrasser, Chem. Phys. Lett. 359, (2002).
dimensional infrared spectroscopy: Studies of the amics of structures with femtosecond pulse Fouriersform correlation spectroscopy. R. M. hstrasser, N. H. Ge, S. Gnanakaran, and M. T. ni, Bull. Chem. Soc. Jap. 75, 1103 (2002).
cts of vibrational frequency correlations on two-ensional infrared spectra. N.-H. Ge, M. T. Zanni, R. M. Hochstrasser, J. Phys. Chem. A 106, 962 2).
temperature dependent structure distribution of a cal peptide studied with 2D IR spectroscopy. M. T. ni, N.-H. Ge, Y. S. Kim, and R. M. Hochstrasser, hys. J. 82, 66 Part 2 (2002).
tosecond two-dimensional infrared spectroscopy: OSY and THIRSTY. N.-H. Ge and R. M. hstrasser, Physchemcomm. 3, U1-U23 (2002).
0A mutant of F TraI relaxase domain: Reduced ity and specificityfor ssDNA and altered rescence anisotropy of a bound labeled onucliotide. M. J. Harley, D. Toptygin, T. Troxler, J. F. Schildbach, Biochemistry 41, 6460 (2002).
TER 13
cofacially compressed, pi-stacked porphyrin-bridge-quinone systems. Y. K. Kang, I. V. Rubtsov, P. M. Iovine, and M. J. Therien, J. Am. Chem. Soc. 124, 8275 (2002).
Synthesis, excited-state dynamics, and reactivity of a directly-linked pyromellitimide –(porphinato)zinc(II) complex. N. P. Redmore, I. V. Rubtsov, and M. R. Therien, Chem. Phys. Lett. 352, 357 (2002).
Energy transfer enhanced quenching in conjugated polymer chemosensors: theory and applications. C. B.Murphy, Y. Zhang, T. Troxler, V. Ferry, and W. E. Jones Jr., Polym. Mat. Sci. Eng. 87, 134 (2002).
Single molecule fluorescence of cytochrome c. E. Mei, J. M. Vanderkooi, and R. M. Hochstrasser, Biophys. J. 82 Part 2, 230 (2002).
Helix formation via conformation diffusion search. C. Y. Huang, Z. Getahun, Y. J. Zhu, J. W. Klemke, W. F. DeGrado, and F. Gai, Proc. Natl. Acad. Sci. USA 99, 2788 (2002).
Conformational preferences and vibrational frequency distributions of short peptides in relation to multidimensional infrared spectroscopy. S. Gnanakaran and R. M. Hochstrasser, J. Am. Chem. Soc. 123, 12886 (2001).
Two-dimensional infrared spectroscopy: A promising new method for the time resolution of structures. M. T. Zanni and R. M. Hochstrasser, Curr. Opin. Struc. Biol. 11, 516 (2001).
Unusual vibrational dynamics of the acetic acid dimer. M. Lim and R. M. Hochstrasser, J. Phys. Chem. 115, 7629 (2001).
Structure and dynamics of proteins and peptides: femtosecond two-dimensional infrared spectroscopy. P. Hamm and R. M. Hochstrasser, Pract. Spectrosc. 26, 273 (2001).
Two-dimensional IR spectroscopy can be designed to eliminate the diagonal peaks and expose on the crosspeaks needed for structure determinatin. M. T. Zanni, N. H. Ge, Y. S. Kim, and R. M. Hochstrasser, Proc. Natl. Acad. Sci. USA, 98, 11265 (2001).
Heterodyned two-dimensional infrared spectroscopy of solvent-dependent conformations of acetylproline-
THE RLBL NEW
NH2. M. T. Zanni, S. Gnanakaran, J. Stenger, and R. M. Hochstrasser, J. Phys. Chem. B 105, 6520 (2001).
Dynamic infrared band-band spectroscopy of peripheral light-harvesting complexes from R. acidophila. R. Kumble, T. D. Howard, R. J. Cogdell, and R. M. Hochstrasser, J. Photochem. Photobiol. A 142, 121 (2001).
Fluorescence lifetime distribution of single molecules undergoing Forster energy transfer. M. Lee, J. Tang and R. M. Hochstrasser, Chem. Phys. Lett. 344, 501 (2001).
Temperature-dependent helix-coil transition of an alanine based peptide. C. Y. Huang, J. W. Klemke, Z. Getahun, W. F. DeGrado, and F. Gai, J. Am. Chem. Soc. 123, 9235 (2001).
Time-resolved infrared study of the helix-coil transition using C-13-labeled helical peptides. C. Y. Huang, Z. Getahun, T. Wang, W. F. DeGrado, F. Gai, J. Am. Chem. Soc. 123, 12111 (2001).
Excitonic behavior in transition metal loaded fluorescent conjugated polymers in solution. C. B. Murphy, Y. Zhang, S. Gilje, T. Troxler, W. E. Jones, Abstr. Pap. Am. Chem. Soc. 222, 524-INOR, Part 1 (2001).
Ultrafast electron transfer in cofacially aligned, π-stacked aromatic systems, II. Y. K. Kang, I. Rubtsov, P. M. Iovine, and M. J. Therien, Abstr. Pap. Am. Chem. Soc. 221, 289-ORGN (2001).
Synthesis, spectroscopy, photophysics of multichromophoric 5,15-bis[[4’-bis(terpyridyl)metal]-ethynylporphinato]zinc(II) assemblies. T. H. Uyeda and M. J. Therien, Abstr. Pap. Am. Chem. Soc. 221, 398-INOR (2001).
Two-dimensional heterodyned and stimulated infrared photon echoes of N-ethylacetamide-D. M. T. Zanni, M. C. Asplund, and R. M. Hochstrasser, J. Chem. Phys. 114, 4579 (2001).
An intense broadband terahertz source based on a novel four wave rectification process. D. J. Cook, J.-X. Chen, and R. M. Hochstrasser, p. 197ff in Ultrafast Phenomena XII: T. Elsaesser, S. Mukamel, M. M. Murane, and N. F. Scherer (eds.), Springer Verlag (2001).
SLETTER 14
Application for Use of the RLBL
Title: Keywords (optional) NIH Axis Numbers (optional) Axis I Axis II Investigators (PI first) Degree Department / Institution / Address 1. 2. 3. NIH Support Sources NIH Start/End Date Other Support Sources Grant Number(s) (MM/DD/YY-MM/DD/YY) Agency and Grant Number(s) 1. 2. Abstract: Describe briefly (200-250 words) the scientific goals and methods. Logistics: Equipment to be supplied by applicant, needed from RLBL, and anticipated time. Telephone Number: FAX Number: E-Mail address: Date: Mailing Address: Send to: Professor R.M. Hochstrasser Director, RLBL Dept. of Chemistry University of Pennsylvania
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Philadelphia, PA 19104-6323
ADDRESS CORRECTION REQUESTED
Mailing Address Street Number and Name City, State 98765-4321
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R L B L c/o T. Troxler Department of Chemistry, UPenn 231S 34th Street Philadelphia, PA 19104-6323
NON-PROFIT ORGAN.U. S. POSTAGE
PAID PERMIT # 2563
PHIILADLEPHIA, 4 PA
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