Pencil excitation with interleaved fourier velocity encoding: NMR measurement of aortic distensibility.

A technique is presented for rapidly and noninvasively determining aortic distensibility, by NMR measurement of pulse-wave velocity in the aorta. A cylinder of magnetization is excited along the aorta, with Fourier-velocity encoding and readout gradients applied along the cylinder axis. Cardiac gating and data interleaving improve the effective time resolution to as high as 3 ms. Wave velocities are determined from the position of the foot of the flow wave in the velocity profiles. Evidence of helical flow distal to the aortic arch can be seen in normal subjects, while disturbed flow patterns are visible in patients with aneurysms and dissections.

Gd3+ vibronic side band spectroscopy. New optical probe of Ca2+ binding sites applied to biological macromolecules.

A new spectroscopic technique is presented for obtaining infraredlike spectra of the binding sites of Ca2+ and other metals in biological macromolecules. The technique, based on the Ca(2+)-like binding properties of Gd3+, utilizes vibronic side bands (VSB) that appear in Gd3+ fluorescence. In the fluorescence spectrum of Gd3+, the separation in photon frequency between a VSB and its electronic origin at approximately 32,150 cm-1 (approximately 311 nm) is a direct measure of the vibrational frequency of a ligand coordinated to Gd3+ ion. As a consequence, the VSB are uncomplicated by molecular vibrations distant from the Gd3+ binding site. The vibrational spectra resulting from the VSB of Gd3+ coordinated to a Ca2+ binding protein, a phospholipid, and DNA are presented.

Carboxy Mb at pH 3. Time-resolved resonance Raman study at cryogenic temperatures.

Cryogenic samples of MbCO at pH3 are studied using nanosecond and picosecond time-resolved resonance Raman spectroscopy. It is observed that under excitation conditions sufficient to completely photodissociate MbCO at pH7, the pH3 sample at 10 ns remains substantially unphotolyzed even at 15 K. The similarity in the optical and resonance Raman spectra of MbCO at pH3 with that of pH7 indicates that at pH3 the iron remains six-coordinate and low-spin. The Fe-CO stretch frequency is consistent with a more upright CO orientation. The absence of the v(Fe-His) band in the 30 ps photoproduct Raman spectrum suggests that the Fe-His(F8) bond is broken within 30 ps of photodissociation. Other Raman bands, though, are not consistent with a normal four-coordinate heme for the photoproduct, Mb*. Suggested possible interpretations include a four-coordinate heme highly perturbed by the close lying protonated proximal histidine or a five-coordinate heme with the Fe-His bond significantly weakened. The partial photolysis monitored at 30 ps and 100 K indicates either a significant amount of geminate recombination within 30 ps or low quantum yield or photolysis. The time course for CO recombination is monitored via the Raman spectra from 30 ps to 3 ns at 100 K and 160 K. Of the fraction of protein-ligand pairs that remain photodissociated at 30 ps, 50% recombine by approximately 250 ps at 100 K and 160 K, supporting the flash photolysis rebinding data of Cowen et al. (Cowen, B. R. 1990. Ph. D. thesis. University of Illinois at Urbana-Champaign; Cowen, B. R., D. Braunstein, H. Frauenfelder, P. J. Steinbach, and R. D. Young. 1989. Biophys. J. 55:55a. [Abstr.].) The conclusions from these resonance Raman studies are extended to solution phase studies at ambient temperatures.

Conformational substates and motions in myoglobin. External influences on structure and dynamics.

Myoglobin, a simppe dioxygen-storage protein, is a good laboratory for the investigation of the connection between protein structure, dynamics, and function. Fourier-transform infrared spectroscopy on carbon-monoxymyoglobin (MbCO) shows three major CO bands. These bands are excellent probes for the investigation of the structure-function relationship. They have different CO binding kinetics and their CO dipoles form different angles with respect to the heme normal, implying that MbCO exists in three major conformational substates, A0, A1, and A3. The entropies and enthalpies of these substates depend on temperature above approximately 180 K and are influenced by pH, solvent, and pressure. These results suggest that even a protein as simple as Mb can assume a small number of clearly different structures that perform the same function, but with different rates. Moreover, protein structure and dynamics depend strongly on the interaction of the protein with its environment.

Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin. The connection between spectral and functional heterogeneity.

The rebinding kinetics of CO to myoglobin after flash photolysis is nonexponential in time below approximately 180 K; the kinetics is governed by a distribution of enthalpic barriers. This distribution results from inhomogeneities in the protein conformation, referred to as conformational substates. Hole-burning experiments on the Soret and IR CO-stretch bands test the assumption that an inhomogeneous distribution of conformational substates results in inhomogeneously broadened spectra. CO was slowly photolyzed at different wavelengths in the Soret band at 10 K. Both the Soret band and the CO-stretch band A1, centered at 1,945 cm-1, shift during photolysis, demonstrating that different wavelengths excite different parts of the distributed population. We have also done kinetic hole-burning experiments by measuring peak shifts in the Soret and A1 bands as the CO molecules rebind. The shifts indicate that the spectral and enthalpic distributions are correlated. In the A1 band, the spectral and enthalpic distributions are highly correlated while in the Soret the correlation is weak. From the peak shifts in the spectral and kinetic hole-burning experiments the inhomogeneous broadening is estimated to be approximately 15% of the total width in the Soret band and approximately 60% in A1. We have previously measured the tilt angle alpha between the bound CO and the heme normal (Ormos, P., D. Braunstein, H. Frauenfelder, M. K. Hong, S.-L. Lin, T. B. Sauke, and R. D. Young. 1988. Proc. Natl. Acad. Sci. USA. 85:8492-8496) and observed a wave number dependence of the tilt angles within the CO-stretch A bands. Thus the spectral and enthalpic distributions of the A bands are coupled to a heterogeneity of the structure.

Charge motion in MbCO crystals after flash photolysis.

Charge motion accompanying the dissociation and recombination of carbon monoxide to oriented myoglobin crystals has been observed. The magnitude of the electrical signals detected after photodissociation by electrodes on either side of MbCO crystals of type A is consistent with the x-ray data showing that the doubly charged iron ion lies in the mean heme plane when a ligand is bound and moves out of the plane when deligated. Beyond 10 ms, the time development of the electrical signal is consistent with the kinetics observed optically after flash photolysis on the same crystals.

Rebinding and relaxation in the myoglobin pocket.

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.

Ligand binding to heme proteins: relevance of low-temperature data.

Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.

Protein states and proteinquakes.

After photodissociation of carbon monoxide bound to myoglobin, the protein relaxes to the deoxy equilibrium structure in a quake-like motion. Investigation of the proteinquake and of related intramolecular equilibrium motions shows that states and motions have a hierarchical glass-like structure.