Gurusamy Balakrishnan

Protein Dynamics from Time-Resolved UV Raman Spectroscopy

Raman spectroscopy can provide unique information on the evolution of structure in proteins over a wide range of time scales; the picosecond to millisecond range can be accessed with pump-probe techniques. Specific parts of the molecule are interrogated by tuning the probe laser to a resonant electronic transition, including the UV transitions of aromatic residues and of the peptide bond. Advances in laser technology have enabled the characterization of transient species at an unprecedented level of structural detail. Applications to protein unfolding and allostery are reviewed.

Tunable kHz Deep Ultraviolet (193–210 nm) Laser for Raman Applications

The performance characteristics of a kilohertz solid-state laser source for ultraviolet Raman spectroscopy are described. Deep ultraviolet (UV) excitation in the 193–210 nm region is provided by mixing of the fundamental and third harmonics of a Ti–sapphire laser, which is pumped by the second harmonic of a Q-Switched Nd–YLF laser. The combination of tunability, narrow linewidth, high average power, good stability, and kilohertz repetition rate makes this laser suitable for deep UV resonance Raman applications. The short pulse duration (∼20 ns) permits nanosecond time resolution in pump–probe applications. The low peak power and high data rate provide artifact-free spectra with a high signal-to-noise ratio. UV Raman cross-section and Raman excitation profiles are reported for gaseous O2 (relative to N2), aqueous ClO4−, tyrosine, phenylalanine, tryptophan, histidine, and hemoglobin excited between 193 nm and 210 nm to illustrate laser performance.

Heme Reactivity is Uncoupled from Quaternary Structure in Gel-Encapsulated Hemoglobin: A Resonance Raman Spectroscopic Study

Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, ν(FeHis), is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving ν(FeHis) frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and reformation of E and F interhelical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.

Differential Control of Heme Reactivity in Alpha and Beta Subunits of Hemoglobin: A Combined Raman Spectroscopic and Computational Study

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or β hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe–histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or β chains, prior to the quaternary R–T and T–R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe–His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem.2002, 98, 149). Experimentally, the νFeHis evolution is faster for β than for α chains, and pump–probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 μs for β but not for α hemes, an interval previously shown to be the first step in the R–T transition. In the α chain νFeHis dropped sharply at 20 μs, the final step in the R–T transition. The time courses are fully consistent with recent computational mapping of the R–T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A.2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.

Temperature-Jump Apparatus with Raman Detection Based on a Solid-State Tunable (1.80–2.05 μm) kHz Optical Parametric Oscillator Laser

The operating characteristics of a pulsed (10 ns) tunable near-infrared (NIR) laser source are described for temperature-jump (T-jump) applications. A Q-switched Nd:YLF laser (∼10 ns pulses) with a 1 kHz repetition rate is used to pump a potassium titanyl arsenate (KTA) crystal-based optical parametric oscillator (OPO), producing ∼1 mJ NIR pulses that are tunable (1.80–2.05 μm) across the 1.9 μm vibrational overtone band of water. This T-jump source has been coupled to a deep ultraviolet (UV) probe laser for Raman studies of protein dynamics. T-jumps of up to 30 °C, as measured via the O–H stretching Raman band of water, are readily achieved. Application to cytochrome c unfolding is demonstrated.

Mode Recognition in UV Resonance Raman Spectra of Imidazole: Histidine Monitoring in Proteins

The imidazole side-chains of histidine residues perform key roles in proteins, and spectroscopic markers are of great interest. The imidazole Raman spectrum is subject to resonance enhancement at UV wavelengths, and a number of UVRR markers of structure have been investigated. We report a systematic experimental and computational study of imidazole UVRR spectra, which elucidates the band pattern, and the effects of protonation and deprotonation, of H/D exchange, of metal complexation, and of addition of a methyl substituent, modeling histidine itself. A consistent assignment scheme is proposed, which permits tracking of the bands through these chemical variations. The intensities are dominated by normal mode contributions from stretching of the strongest ring bonds, C(2)N and C(4)C(5), consistent with enhancement via resonance with a dominant imidazole π-π* transition.

Subunit-Selective Interrogation of CO Recombination in Carbonmonoxy Hemoglobin by Isotope-Edited Time-Resolved Resonance Raman Spectroscopy

Hemoglobin (Hb) is an allosteric tetrameric protein made up of alphabeta heterodimers. The alpha and beta chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with (15)N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For alpha but not for beta chains, the frequency of the nu(4) porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the alpha chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 +/- 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the alphabeta interface, evidence for which has emerged from UVRR studies of site mutants.

Ultrafast Charge Transfer in Nickel Phthalocyanine Probed by Femtosecond Raman-Induced Kerr Effect Spectroscopy

The recently developed technique of femtosecond stimulated Raman spectroscopy, and its variant, femtosecond Raman-induced Kerr effect spectroscopy (FRIKES), offer access to ultrafast excited-state dynamics via structurally specific vibrational spectra. We have used FRIKES to study the photoexcitation dynamics of nickel(II) phthalocyanine with eight butoxy substituents, NiPc(OBu)8. NiPc(OBu)8 is reported to have a relatively long-lived ligand-to-metal charge-transfer (LMCT) state, an essential characteristic for efficient electron transfer in photocatalysis. Following photoexcitation, vibrational transitions in the FRIKES spectra, assignable to phthalocyanine ring modes, evolve on the femtosecond to picosecond time scales. Correlation of ring core size with the frequency of the ν10 (asymmetric C–N stretching) mode confirms the identity of the LMCT state, which has a ∼500 ps lifetime, as well as that of a precursor d-d excited state. An even earlier (∼0.2 ps) transient is observed and tentatively assigned to a higher-lying Jahn–Teller-active LMCT state. This study illustrates the power of FRIKES spectroscopy in elucidating ultrafast molecular dynamics.

Quaternary Speeding in Hemoglobin

Fig. 1. Hb ‘hinge’ and ‘switch’ contacts formed at the α1β2 interface by the R–T dimer rotation. 7 How fast can proteins rearrange their quaternary structure? The answer seems to be very fast indeed. A time constant of only 1.2 μs has been reported for ion channel opening in the multimeric membrane proteinAChR. And in this issue of JMB,Cammarata et al. report 2 μs as the time constant for the R–T quaternary transition in hemoglobin (Hb), whereas 20 μs had long been the accepted value. Gibsons classic study of the recombination kinetics following photodissociation of the CO adduct, HbCO, revealed fast and slow phases with a 20-μs transition between them. Subsequently, Hofrichter et al. also obtained 20 μs as the last relaxation of the deoxy-heme absorption spectrum, subsequent to HbCO photodissociation, and assigned it to the quaternary transition on the basis of its dependence on the degree of photolysis. However, the time course of ultraviolet resonance Raman (UVRR) spectra, which select for vibrational signals of tyrosine and tryptophan residues, has revealed that the R–T transition is actually a twostage process. From crystallography, it is known that the change in quaternary structure involves a 15° rotation of one αβ dimer against the other (see Fig. 1) and results in substantial realignment of the α1β2 intersubunit interface. There are two main contact alterations. One is at the ‘hinge’, where the contacting side chains reorient, and an H-bond forms from Trpβ37 to Aspα94 in the T structure. The other is at the ‘switch’, where the rotation switches the registry of the contacting groups, and an H-bond forms between Tyrα42 and Aspβ99, again in the T structure. The UVRR difference spectrum between HbCO and deoxyHb contains signatures for both of these H-bonds, and pumpprobe difference spectra following HbCO photodissociation revealed that they form in sequence. The ‘hinge’ contact forms in 2 μs, while the ‘switch’ contact forms in 20 μs, coincident with the previously accepted R–T transition time. It was pointed out that formation of the Trpβ37-Aspα94 H-bond implies substantial quaternary motion at 2 μs, but the extent of the motion could not be determined from the spectral response. A 2-μs time constant for the Trpβ37 H-bond was also reported from a timeresolved circular dichroism study, but again the structural implication was uncertain. Cammarata et al. usedwide-angle X-ray scattering (WAXS) to monitor the response to HbCO photodissociation. WAXS gives averaged structural infor-

Temperature-Jump Fluorescence Provides Evidence for Fully Reversible Microsecond Dynamics in a Thermophilic Alcohol Dehydrogenase.

Protein dynamics on the microsecond (μs) time scale were investigated by temperature-jump fluorescence spectroscopy as a function of temperature in two variants of a thermophilic alcohol dehydrogenase: W87F and W87F:H43A. Both mutants exhibit a fast, temperature-independent μs decrease in fluorescence followed by a slower full recovery of the initial fluorescence. The results, which rule out an ionizing histidine as the origin of the fluorescence quenching, are discussed in the context of a Trp49-containing dimer interface that acts as a conduit for thermally activated structural change within the protein interior.