Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

The photoisomerization of retinal in bacteriorhodopsin: Experimental evidence for a three-state model

The primary events in the all-trans to 13-cis photoisomerization of retinal in bacteriorhodopsin have been investigated with femtosecond time-resolved absorbance spectroscopy. Spectra measured over a broad range extending from 7000 to 22,400 cm−1 reveal features whose dynamics are inconsistent with a model proposed earlier to account for the highly efficient photoisomerization process. Emerging from this work is a new three-state model. Photoexcitation of retinal with visible light accesses a shallow well on the excited state potential energy surface. This well is bounded by a small barrier, arising from an avoided crossing that separates the Franck–Condon region from the nearby reactive region of the photoisomerization coordinate. At ambient temperatures, the reactive region is accessed with a time constant of ≈500 fs, whereupon the retinal rapidly twists and encounters a second avoided crossing region. The protein mediates the passage into the second avoided crossing region and thereby exerts control over the quantum yield for forming 13-cis retinal. The driving force for photoisomerization resides in the retinal, not in the surrounding protein. This view contrasts with an earlier model where photoexcitation was thought to access directly a reactive region of the excited-state potential and thereby drive the retinal to a twisted conformation within 100–200 fs.

Three-dimensional model of the potyviral genome-linked protein.

The full sequence of the genome-linked viral protein (VPg) cistron located in the central part of potato virus Y (common strain) genome has been identified. The VPg gene codes for a protein of 188 amino acids, with significant homology to other known potyviral VPg polypeptides. A three-dimensional model structure of VPg is proposed on the basis of similarity of hydrophobic-hydrophilic residue distribution to the sequence of malate dehydrogenase of known crystal structure. The 5' end of the viral RNA can be fitted to interact with the protein through the exposed hydroxyl group of Tyr-64, in agreement with experimental data. The complex favors stereochemically the formation of a phosphodiester bond [5'-(O4-tyrosylphospho)adenylate] typical for representatives of picornavirus-like viruses. The chemical mechanisms of viral RNA binding to VPg are discussed on the basis of the model structure of protein-RNA complex.

The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli.

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8, 3933-3938]. The alternative "allosteric three-site model" [Nierhaus, K.H. (1990) Biochemistry 29, 4997-5008] features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner. Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA. The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines. At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G. On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable. Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.

A functionally defined model for the M2 proton channel of influenza A virus suggests a mechanism for its ion selectivity

The M2 protein from influenza A virus forms proton-selective channels that are essential to viral function and are the target of the drug amantadine. Cys scanning was used to generate a series of mutants with successive substitutions in the transmembrane segment of the protein, and the mutants were expressed in Xenopus laevis oocytes. The effect of the mutations on reversal potential, ion currents, and amantadine resistance were measured. Fourier analysis revealed a periodicity consistent with a four-stranded coiled coil or helical bundle. A three-dimensional model of this structure suggests a possible mechanism for the proton selectivity of the M2 channel of influenza virus.

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.

Muscle protein synthesis by positron-emission tomography with l-[methyl-11C]methionine in adult humans

Existing methods for assessing protein synthetic rates (PSRs) in human skeletal muscle are invasive and do not readily provide information about individual muscle groups. Recent studies in canine skeletal muscle yielded PSRs similar to results of simultaneous stable isotope measurements using l-[1-13C, methyl-2H3]methionine, suggesting that positron-emission tomography (PET) with l-[methyl-11C]methionine could be used along with blood sampling and a kinetic model to provide a less invasive, regional assessment of PSR. We have extended and refined this method in an investigation with healthy volunteers studied in the postabsorptive state. They received ≈25 mCi of l-[methyl-11C]methionine with serial PET imaging of the thighs and arterial blood sampling for a period of 90 min. Tissue and metabolite-corrected arterial blood time activity curves were fitted to a three-compartment model. PSR (nmol methionine⋅min−1⋅g muscle tissue−1) was calculated from the fitted parameter values and the plasma methionine concentrations, assuming equal rates of protein synthesis and degradation. Pooled mean PSR for the anterior and posterior sites was 0.50 ± 0.040. When converted to a fractional synthesis rate for mixed proteins in muscle, assuming a protein-bound methionine content of muscle tissue, the value of 0.125 ± 0.01%⋅h−1 compares well with estimates from direct tracer incorporation studies, which generally range from ≈0.05 to 0.09%⋅h−1. We conclude that PET can be used to estimate skeletal muscle PSR in healthy human subjects and that it holds promise for future in vivo, noninvasive studies of the influences of physiological factors, pharmacological manipulations, and disease states on this important component of muscle protein turnover and balance.

Structural determinants of oxidative folding in proteins

A method for determining the kinetic fate of structured disulfide species (i.e., whether they are preferentially oxidized or reshuffle back to an unstructured disulfide species) is introduced. The method relies on the sensitivity of unstructured disulfide species to low concentrations of reducing agents. Because a structured des species that preferentially reshuffles generally first rearranges to an unstructured species, a small concentration of reduced DTT (e.g., 260 μM) suffices to distinguish on-pathway intermediates from dead-end species. We apply this method to the oxidative folding of bovine pancreatic ribonuclease A (RNase A) and show that des[40–95] and des[65–72] are productive intermediates, whereas des[26–84] and des[58–110] are metastable dead-end species that preferentially reshuffle. The key factor in determining the kinetic fate of these des species is the relative accessibility of both their thiol groups and disulfide bonds. Productive intermediates tend to be disulfide-secure, meaning that their structural fluctuations preferentially expose their thiol groups, while keeping their disulfide bonds buried. By contrast, dead-end species tend to be disulfide-insecure, in that their structural fluctuations expose their disulfide bonds in concert with their thiol groups. This distinction leads to four generic types of oxidative folding pathways. We combine these results with those of earlier studies to suggest a general three-stage model of oxidative folding of RNase A and other single-domain proteins with multiple disulfide bonds.

Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine.

Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.

Folding thermodynamics of a model three-helix-bundle protein

The calculated folding thermodynamics of a simple off-lattice three-helix-bundle protein model under equilibrium conditions shows the experimentally observed protein transitions: a collapse transition, a disordered-to-ordered globule transition, a globule to native-state transition, and the transition from the active native state to a frozen inactive state. The cooperativity and physical origin of the various transitions are explored with a single “optimization” parameter and characterized with the Lindemann criterion for liquid versus solid-state dynamics. Below the folding temperature, the model has a simple free energy surface with a single basin near the native state; the surface is similar to that calculated from a simulation of the same three-helix-bundle protein with an all-atom representation [Boczko, E. M. & Brooks III, C. L. (1995) Science 269, 393–396].