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B. Coupling of Protein Fluctuations to the CO Ligand at the Active Site

B. Coupling of Protein Fluctuations to the CO Ligand at the Active Site

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energy. Two models have been proposed to explain the dephasing in Mb

(20,102). One involves global electric field fluctuations and the other local

mechanical coupling.

In the global electric field model, motions of polar groups throughout

the protein produce a time-dependent electric field. The fluctuating electric

field causes modulation of the electron density of the heme’s delocalized

-electron cloud. Fluctuations of the heme

electron density modulate

the magnitude of the back bonding to the CO Ł orbital, causing timedependent shifts in CO , or pure dephasing. In essence, the protein acts as a

fluctuating electric field transmitter. The heme acts like an antenna, which

receives the signal of protein fluctuations and communicates it to the CO

ligand bound at the active site via the back bonding.

In the local mechanical fluctuation model, the local motions of the

amino acids on the proximal side of the heme are coupled to the heme

through the side group of the proximal histidine. The side chain of the

proximal histidine is covalently bonded to the Fe. This bond is the only

covalent bond of the heme to the rest of the protein. Thus, motions of

the ˛-helix that contains the proximal histidine are directly coupled the Fe.

These motions can push and pull the Fe out of the plane of the heme. Since

the CO is bound to the Fe, these motions may induce changes in the CO

vibrational transition frequency causing pure dephasing.

To test these models, we have performed a temperature-dependent

vibrational echo and pump-probe study on two myoglobin mutants, H64VCO and H93G(N-MeIm)-CO, and compared the results to those of the

wild-type protein. To test the global electric field model, we studied H64V,

a myoglobin mutant in which the polar distal histidine is replaced by a

nonpolar valine (105). If the global electric field model of the dephasing

is operative, then the decrease in the electric field in the mutant should

reduce the magnitude of the frequency fluctuations, producing slower pure

dephasing. To test the local mechanical model of pure dephasing, we studied

H93G(N-MeIm), a myoglobin mutant in which the proximal histidine is

replaced by a glycine (106). This mutation severs the only covalent bond

between the heme and the globin and leaves a large open pocket on the

proximal side of the heme. Inserted into this pocket and bound to the heme

at the Fe is an exogenous N -methylimidizole, which has similar chemical

and electrostatic properties as the side group of the histidine. Effectively,

the proximal bond has been severed without changing significantly the electrostatic properties of the protein. If dynamics of the f ˛-helix are causing

the pure dephasing by producing Fe motions via the proximal histidine, then

the dephasing of this mutant should be less than that of the native protein.



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Rector and Fayer



Figure 14 Pure dephasing rate versus temperature for native MbCO in ethylene glycol/H2 O (circles, same as in Fig. 13). Also plotted is pure dephasing for

two mutants of myoglobin, H64V-CO (squares) and H93G(N-MeIm)-CO (triangles). The native MbCO and the mutant H93G(N-MeIm)-CO have identical pure

dephasing temperature dependences. The H64V-CO has identical form of the pure

dephasing but with a 21 š 3% decrease in the pure dephasing rate at all temperatures

studied.



Figure 14 shows the pure dephasing rates versus temperature on a log

plot of the wild-type protein and the two mutants studied, all in 95:5% glycerol:water (102). The circles are the values for the native protein, which are

the same as in Fig. 13. The triangles are the mutant H93G(N-MeIm)-CO

pure dephasing rates. Clearly these values are identical to the native protein

within experimental error, indicating that the proposed local mechanical

dephasing model is not active in myoglobin. The squares are the pure

dephasing rates for the mutant H64V-CO in 95:5% glycerol:water. The

data have the same temperature dependence as the wild type. However, the

dephasing is 21 š 3% slower than that of the wild type at all temperatures.

The functional form of the temperature dependence is unchanged because



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Infrared Vibrational Echo Experiments



265



modification of one amino acid does not significantly change the global

dynamics of the protein. However, replacing the polar distal histidine with

a nonpolar valine removes one source of the fluctuating electric fields,

reduces the coupling of the protein dynamics to the CO vibration, and

slows dephasing. These results support the global electric field model of

pure dephasing in myoglobin and suggest that the distal histidine contributes

21 š 3% of the fluctuating electric fields felt at the heme. Recent molecular dynamics simulations (107) lend support to the ¾20% electric field

fluctuation produced by the distal histidine.

Figure 15 shows pure dephasing of Hb-CO in EgOH/H2 O, performed

with the OPA and a comparison to MbCO in the same solvent mixture. The

line through the MbCO data is the same line as in Fig. 13. The line through

the Hb-CO data is the same as the Mb line multiplied by 0.73. On a log plot

multiplication by a constant corresponds to a linear shift. It is clear from

the data that the functional forms of the data on Mb and Hb are identical.

This suggests that both the low-temperature TLS dynamics and the hightemperature combination Arrhenius and viscosity dependences active in Mb

are also active in Hb. Considering the implications of Fig. 12, it is possible

that the difference in the pure dephasing rates in the two proteins is caused

by differences in the fluctuating electric field magnitudes felt at the heme in

the two proteins. This proposal would suggest that the fluctuating electric

field magnitude felt at the heme and coupled to the CO is 27% lower in

Hb than in Mb. Qualitative agreement with this concept has been seen in

mutant protein studies (107). The nature of the dephasing in MbCO and

HbCO is under continuing experimental and theoretical study.

VI. CONCLUDING REMARKS



Vibrational echo experiments have made it possible to perform a detailed

examination of the dynamics of inter- and intramolecular interactions that

give rise to the homogeneous linewidths and pure dephasing of the asymmetric CO-stretching mode of Rh(CO)2 acac in liquid and glassy solvents

and of the stretching mode of CO bound at the active site of the globin

proteins, myoglobin and hemoglobin. At low temperature (3.5 to ¾80 K),

Rh(CO)2 acac temperature-dependent pure dephasing has the function form,

T1 . This is interpreted as the result of coupling between the vibrational

mode and the dynamical two-level systems of the glassy DBP solvent.

Above ¾80 K, the pure dephasing becomes exponentially activated with

an activation energy of ¾400 cm 1 . There is no change in the functional

form of the temperature dependence in passing from the glass to the liquid.



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Rector and Fayer



Figure 15 Pure dephasing rate of MbCO and HbCO both in ethylene glycol/H2 O

versus temperature. The MbCO data (squares) are the same as those shown in

Fig. 13. The HbCO data (diamonds) have the same functional form of the temperature dependence, but the dephasing is consistently slower at all temperatures.



These results suggest that the activated process arises from coupling of the

high-frequency CO stretch to the internal 405 cm 1 Rh-C asymmetrical

stretching mode.

The vibrational echo spectrum method was demonstrated theoretically

and experimentally. In the VES technique, the delay between the two pulses

in the vibrational echo pulse sequence is fixed and the laser frequency is

scanned across the transition of interest. The VES technique can selectively



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Infrared Vibrational Echo Experiments



267



remove unwanted spectral features, such as a broad background absorption

or undesired peaks, using either differences in homogeneous dephasing

or transition dipole moments. The method was demonstrated on a mixture

Rh(CO)2 acac and W(CO)6 in DBP and on myoglobin-CO near 1950 cm 1 .

Peak selectivity was clearly demonstrated in the inorganic system, and high

optical density background suppression was demonstrated in the protein

system.

Vibrational echo experiments have also been applied to the COstretching mode of myoglobin-CO, mutant myoglobins, and hemoglobinCO. Temperature-dependent vibrational echo and lifetime measurements

have been performed on CO bound to the active site of native Mb in

a variety of solvents and two mutants of myoglobin and HbCO in the

same solvent as a Mb study. In addition, an isothermal (300 K) viscosity

dependence of MbCO has been recorded.

Temperature-dependent pure dephasing rates of MbCO in three

solvents show identical power law behavior at low temperatures. At

intermediate temperatures there is a break in the power law arising from

the solvent-influenced protein glass transition. Above this point the data

in glassy trehalose are exponentially activated. The other solvents, which

at elevated temperatures are liquids, have additional solvent viscositydependent contributions to the pure dephasing rate.

The temperature-dependent vibrational echo results show that the pure

dephasing of H64V is ¾21 š 3% slower than native Mb, with no change

in the functional form of the temperature dependence. The temperature

dependence of the pure dephasing of H93G(N-MeIm) is identical to the

native Mb. The general mechanism proposed (16) to explain the coupling

of conformational fluctuations of the protein to the vibrational transition

energy of CO bound at the active site is supported by the H64V results.

The model states that protein motions produce fluctuating electric fields,

which are responsible for the CO pure dephasing. Replacing the polar distal

histidine with the nonpolar valine removes one source of the fluctuating

electric fields, thus reducing the coupling between the protein fluctuations

and the measured pure dephasing. The picture that emerges is that the heme

acts as an antenna that receives and then communicates protein fluctuations

to the CO ligand bound at the active site.

The vibrational echo results on HbCO in EgOH/H2 O show an identical functional form of the temperature dependence as MbCO in the same

solvent mixture. We therefore concluded that the same dephasing mechanisms active in Mb are also active in Hb. In both proteins, a power law, T1.3 ,

is observed at low temperatures. This temperature dependence, observed in



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two proteins, strongly suggests that the protein behaves in a dynamical

manner that is similar to a glass at low temperatures. The magnitude of the

pure dephasing is 27% less in Hb than in Mb, suggesting that the magnitude

of the electric field fluctuations is lower by this amount in Hb.

The ultrafast infrared vibrational echo experiment and vibrational

echo spectroscopy are powerful new techniques for the study of molecules

and vibrational dynamics in condensed matter systems. In 1950, the advent

of the NMR spin echo (1) was the first step on a road that has led to

the incredibly diverse applications of NMR in many fields of science and

medicine. Although vibrational spectroscopy has existed far longer than

NMR, the experiments described here are the first ultrafast IR vibrational

analogs of pulsed NMR methods. In the future, it is anticipated that the

vibrational echo will be extended to an increasingly diverse range of problems and that the technique will be expanded to new pulse sequences,

including multidimensional coherent vibrational spectroscopies such as the

vibrational echo spectroscopy technique describe above.

ACKNOWLEDGMENTS



A large number of individuals participated in the work discussed in this

review. We would like to thank Dr. Alfred Kwok, Dr. Camilla Ferrente,

Dr. David Thompson, Kusai Merchant, Dr. David Zimdars, Dr. Rick

Francis, Stanford University, and Prof. Dana Dlott and Dr. Jeffrey Hill,

University of Illinois at Urbana-Champaign for significant contributions.

We would also like to thank Professors Alan Schwettman and Todd

Smith and their research groups, especially Dr. Christopher Rella and

Dr. James Engholm, at the Stanford Free Electron Laser Center, whose

efforts made these experiments possible. We thank Prof. Stephen Boxer,

Stanford University, and Prof. Steven Sligar, University of Illinois at

Urbana-Champaign, for providing the protein mutants, H93G(N-Melm)

and H64V, respectively. This research was supported by the Office of

Naval Research, (N00014-92-J-1227-P00006, N00014-94-1-1024) and the

National Science Foundation, Division of Materials Research (DMR9322504, DMR-9610326)

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7

Structure and Dynamics of Proteins

and Peptides: Femtosecond

Two-Dimensional Infrared

Spectroscopy

Peter Hamm

Max-Born Institut, Berlin, Germany



Robin M. Hochstrasser

University of Pennsylvania, Philadelphia, Pennsylvania



I. INTRODUCTION



A complete and predictive understanding of biological processes will

require descriptions of the structures, and the dynamics. Knowing the

three-dimensional structures of peptides and proteins is necessary in order

to understand the selectivity and specificity of biological reactions. Xray diffraction and nuclear magnetic resonance NMR spectroscopy (1–4)

are extremely powerful spectroscopic tools with the ability to determine

structures of proteins with hundreds (NMR) or even thousands (x-ray)

of amino acids. The tremendous progress in understanding biological

reactions of all types has been made possible by the detailed knowledge

of the secondary, tertiary, and quaternary structures of the participating

biomolecules. X-ray and NMR studies can also provide information on the

distributions of structures and whether portions of the molecule are partially

or fully disordered. However, the time scales of the fluctuations within

ordered or disordered structures are not so readily obtained. Vibrational



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