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R Tuma

Publications and source records attributed to R Tuma.

34 records · Page 2Linked to original sources
PubMed1998

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.

Binding Sites↗
PubMed1997

Mechanisms of virus assembly probed by Raman spectroscopy: the icosahedral bacteriophage P22.

A microdialysis flow cell has been developed for time-resolved Raman spectroscopy of biological macromolecules and their assemblies. The flow cell permits collection of Raman spectra concurrent with the efflux of small solute molecules into a solution of macromolecules and facilitates real-time spectroscopic detection of structural transitions induced by the effluent. Additionally, the flow cell is well suited to the investigation of hydrogen-isotope exchange phenomena that can be exploited as dynamic probes of viral protein folding and solvent accessibility along the assembly pathway. Here, we describe the application of the Raman dynamic probe to the maturation of the icosahedral capsid of bacteriophage P22, a double-stranded DNA virus. The P22 virion is constructed from a capsid precursor (procapsid) consisting of 420 coat subunits (gp5) in an outer shell and a few hundred scaffolding subunits (gp8) within. Capsid maturation involves expulsion of scaffolding subunits coupled with shell expansion at the time of DNA packaging. Raman static and dynamic probes reveal that the scaffolding subunit is highly alpha-helical and highly thermolabile, and lacks a typical hydrophobic core. When bound within the procapsid, the alpha-helical fold of gp8 is thermostabilized; however, this stabilization confers no apparent protection against peptide NH-->ND exchange. A molten globule model is proposed for the native scaffolding subunit that functions in procapsid assembly. Accompanying capsid expansion, a small conformational change (alpha-helix-->beta-strand) is also observed in the coat subunit. Domain movement mediated by hinge bending is proposed as the mechanism of capsid expansion. On the basis of these results, a molecular model is proposed for assembly of the P22 procapsid.

Bacteriophage P22↗
PubMed1997

A comparison of donor and recipient site sensation in free tissue reconstruction of the oral cavity.

In patients who undergo oral cavity reconstruction, loss of sensation plays a vital role in producing disturbances in postoperative oral function. Microsurgical techniques have provided a method of addressing this deficit through the use of sensate cutaneous free flaps in which microneural anastomoses are performed between a sensory nerve supplying the flap, and a recipient nerve in the head and neck. The purpose of this study was to compare the cutaneous sensation of the radial forearm flap and lateral arm flap donor sites, the two most commonly used intraoral sensate flaps. For comparison, sensation was also determined in five intraoral sites: the tip of tongue, lateral tongue, cheek, gingiva, and hard palate. Sensation was evaluated at the two potential donor sites in 66 random subjects using static and moving two-point discrimination, thermal sensation differences, and Semmes-Weinstein monofilament pressures. In the same subjects Semmes-Weinstein monofilament pressures were used to evaluate intraoral sensation. Information was recorded on age, sex, smoking and denture status. All four sensory evaluations demonstrated that the lateral arm flap donor site was more sensitive than the radial forearm donor site. Thermal sensitivity differentials (0.52 vs. 0.40 degrees C, p < 0.001), static two-point discrimination (15.4 vs. 15.0 mm, p < 0.2), moving two-point discrimination (5.8 vs. 4.8 mm, p < 0.03), and Semmes-Weinstein monofilament pressures (5.10 vs. 4.08 g per square millimeter, p < 0.001) all indicated a more sensitive lateral arm flap donor site. Older subjects had significantly decreased sensation at both donor sites based on static two-point discrimination and Semmes-Weinstein monofilament testing. No sex differences were noted. Based on Semmes-Weinstein monofilament testing in the mouth, the tip of the tongue is the most sensitive area (2.26 g per square millimeter), followed by the hard palate (3.60 g per square millimeter), the lateral tongue (4.08 g per square millimeter), the cheek (4.77 g per square millimeter), and the gingiva (8.06 g per square millimeter). Smokers had significantly decreased sensation at the tip of tongue and hard palate. Denture wearers had significantly diminished sensation in all intraoral locations except the lateral tongue. Older patients had significantly diminished sensation at all intraoral sites. No sex differences were noted. The lateral arm flap donor site is a more sensitive region than the radial forearm flap donor site. However, the lateral arm flap donor site is less sensitive than the tip of tongue and hard palate, while the radial forearm flap donor site is less sensitive than the tip of tongue, hard palate, lateral tongue, and cheek. This suggests that for certain locations, intraoral sensate flaps may require measures such as sensory reeducation protocols to approach normal recipient site sensation.

Adult↗
PubMed1996

Structural transitions in the scaffolding and coat proteins of P22 virus during assembly and disassembly.

An in vitro system for investigating the assembly of the Salmonella phage P22 has been exploited to elucidate the structural basis of recognition between scaffolding protein (gp8) and coat protein (gp5) subunits of the viral procapsid. Raman spectroscopy and circular dichroism have been employed to examine structural thermostabilities of both gp8 and gp5 in native procapsids, and to characterize structural changes accompanying scaffolding exit, procapsid expansion, and shell disassembly. It is found that the secondary structure of the isolated gp8 subunit is rich in alpha-helix (approximately 40%), is highly thermolabile, and is characterized by noncooperative unfolding (Tm approximately 49 degrees C). Conversely, the procapsid-bound gp8 subunit exhibits stabilization of its alpha-helical secondary structure, characterized by cooperative unfolding. Because cooperative unfolding of gp8 coincides with exit from the procapsid, the present results suggest that unfolding and release are coupled processes. Structural differences between procapsid-free and procapsid-bound gp8 subunits are also apparent in Raman markers which monitor environments of tyrosine and tryptophan side chains. Temperature-resolved Raman spectroscopy of the empty procapsid shell reveals three distinct structural transitions for the gp5 subunits. The first, which occurs between 50 and 65 degrees C, is attributed to shell expansion and results in an increase in beta-strand secondary structure. The two higher temperature transitions, occurring within intervals of 70-80 and 80-95 degrees C, respectively, are attributed to partial unfolding of the shell subunit and subsequent shell disassembly. The same gp5 structure transitions are detected for procapsids which contain scaffolding protein. On the basis of the observed thermodynamic coupling between gp8 unfolding and its release from the procapsid, we propose a model for P22 procapsid assembly. Implications of the model for in vivo assembly of dsDNA viruses are discussed.

Bacteriophage P22↗
PubMed1996

Structure, interactions and dynamics of PRD1 virus I. Coupling of subunit folding and capsid assembly.

Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome. The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits. Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation. However, the products of thermal disassembly depend sensitively upon total protein concentration. Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed. Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results. Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C. Thus, shell disassembly diminishes P3 thermostability. Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand. Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration. Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell. The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access. We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit.

Bacteriophages↗
PubMed1996

Structure, interactions and dynamics of PRD1 virus II. Organization of the viral membrane and DNA.

Structure, dynamics and stability of the membrane and double-stranded (ds) DNA genome packaged within the native PRD1 virion have been probed by laser Raman spectroscopy. The Raman signature of PRD1 is complex, but exhibits distinctive marker bands diagnostic of the internal lipid bilayer and dsDNA. The Raman markers demonstrate, respectively, a liquid crystalline lipid phase(L(alpha)) and B DNA conformation throughout the temperature range (5 degrees to 50 degrees) of virion stability. Despite the absence of large scale lipid phase transitions or DNA melting, small temperature-dependent changes in the Raman markers of lipid and DNA are detected, indicating coupling between their structures. Minor deviations of DNA from the canonical B form are imposed by the membrane. The Raman markers indicate further that base stacking and phosphate group interactions of the packaged PRD1 genome differ from those of unpackaged PRD1 DNA. Specific Raman band perturbations are proposed as indicators of DNA-membrane interaction. Hydrogen-deuterium exchange kinetics of packaged and unpackaged PRD1 DNA are indistinguishable, demonstrating that base imino and amino protons are not affected significantly by either the condensation or membrane enclosure associated with DNA packaging. This contrasts with the significant acceleration of base exchanges detected in the packaged DNA of bacteriophage P22, which lacks a viral membrane. The distinctive H-->2H exchange profile of the PRD1 genome, the absence of packaging-induced acceleration of exchange kinetics and the apparent direct interaction between DNA and phospholipids suggest a specific role for the viral membrane in PRD1 assembly. We propose a "membrane-surface-catalyzed" model for dsDNA condensation and organization within the PRD1 virion.

Bacteriophages↗
PubMed1996

Theory, design, and characterization of a microdialysis flow cell for Raman spectroscopy.

The theory, design, and application of a dialysis flow cell for Raman spectroscopy are described. The flow cell permits rapid collection of Raman spectra concurrent with the efflux of small solute molecules or ions into a solution of macromolecules and is well suited to acquisition of data during hydrogen-isotope exchange reactions of biological molecules. Kinetic parameters of the device are described by a diffusion model, which accounts satisfactorily for the observed rates of efflux of deuterium oxide (K2H = 0.30 min-1), calcium ions (KCa = 0.10 min-1) and EGTA (KEGTA = 0.07 min-1). Application to the kinetics of glutamate protonation in a peptide copolymer [poly(Glu, Lys, Tyr)] shows that pH-titration rates as high as 3.3 pH units/min can be monitored. It is also shown that one can extract first-order hydrogen-isotope exchange rate constants from measured second-order exchanges by taking into account the rate of entry of 2H2O effluent into the bulk H2O solution. Deuterium exchanges of the single-stranded polyribonucleotides poly(rA) and poly(rU) and of the double-stranded RNA genome from bacteriophage phi 6 have been investigated. The measured nucleotide base exchange rates are comparable with those determined previously by other methods. The results indicate that base exchanges as fast as approximately 2 min-1 can be determined reliably with the present design. Application of the Raman flow cell to hydrogen-isotope exchange of the basic pancreatic trypsin inhibitor confirms consistency with results obtained previously on this protein by tritiation and NMR techniques.

Calcium↗
PubMed1995

Solution conformation of the extracellular domain of the human tumor necrosis factor receptor probed by Raman and UV-resonance Raman spectroscopy: structural effects of an engineered PEG linker.

The solution structure of the Escherichia coli-expressed extracellular domain, residues 12-172, of the human 55 kDa type I tumor necrosis factor receptor (TNFR) has been probed by Raman (514.5 nm) and ultraviolet-resonance Raman (244 nm) excitations. The Raman spectra have been collected from both the free TNFR domain and an engineered "dumbbell-like" derivative, consisting of two mutant receptor moieties linked by a 20 kDa polyethylene glycol (PEG) tether. The results demonstrate a TNFR secondary structure which is rich in beta-sheet and deficient in alpha-helix, consistent with the reported X-ray crystal structure of baculovirus expressed receptor complexed with factor beta [Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C., Loetscher, H., & Lesslauer, W. (1993) Cell 73, 431-445]. Conversely, the solution structure of TNFR differs from the crystal structure in its distribution of disulfide rotamers and in the orientation of its unique indole side chain (tryptophan-107). These differences are attributed, respectively, to N-terminal truncation and factor binding in the TNFR crystal structure. The tryptophan configuration, which is easily monitored in both Raman and UVRR spectra, is proposed as a potential signal of receptor/factor recognition and binding. Application of the Raman probes to the engineered TNFR dumbbell, which is of interest as a potential therapeutic, shows that TNFR moieties of the dumbbell exhibit secondary structures and side chain environments which are indistinguishable from those of the native, wild-type moiety. The results suggest that the PEGylated dumbbell may function as an effective TNFR drug delivery system without the consequence of a deleterious antigenic response.

Amino Acid Sequence↗
PubMed1994

Purification of viruses and macromolecular assemblies for structural investigations using a novel ion exchange method.

We describe a novel ion exchange chromatographic technique suitable for large-scale preparation of viruses and other biomacromolecular assemblies in highly purified form. The method, which utilizes anion exchange on either of two commercially available cellulose cartridges, is applied to the Escherichia coli bacteriophage PRD1. Viral particles eluted from both QMA and DEAE cartridges retain infectivity and exhibit greater homogeneity of composition, as judged by gel electrophoresis and electron microscopy, than particles purified by rate zonal sucrose gradient centrifugation. The ion exchange protocols are rapid, requiring less than 15 min elution time, and permit retrieval of the purified viral particles at high concentration in aqueous media without centrifugal pelleting. The present method is particularly well suited to the preparation of milligram to decigram quantities of virus, sufficient for many biophysical structural analyses, including investigations by solution spectroscopic and crystal diffraction techniques. The feasibility and advantages of the ion exchange chromatographic procedure are demonstrated by application of laser Raman spectroscopy to ion exchange purified PRD1 virions and subviral assemblies.

Centrifugation, Zonal↗
PubMed1993

Cysteine conformation and sulfhydryl interactions in proteins and viruses. 3. Quantitative measurement of the Raman S-H band intensity and frequency.

The bond stretching vibration of the cysteine sulfhydryl (SH) group in a typical protein generates a Raman band in the spectral interval 2500-2600 cm-1, a region devoid of interference from any other fundamental mode of vibration of the protein. The relatively high Raman cross section associated with the S-H stretching vibration, the sensitivity of the vibrational frequency to hydrogen bonding interactions and side chain configurations, and the dependence of the Raman intensity on thiol-thiolate equilibria, combine to make the Raman SH band a potentially valuable marker of protein sulfhydryl interactions and a unique indicator of sulfhydryl participation in thiol-disulfide oxidoreductase activity. In order to exploit Raman spectroscopy for these purposes, accurate and precise measurements of Raman SH band profiles are required. We show here that the required precision and accuracy can be achieved by use of the Raman band corresponding to the stretching vibration of in situ nitrogen gas as a quantitative intensity and frequency standard. The Raman Q-branch center of the N2 band occurs at 2330.7 cm-1.

Bacteriophage P22↗
PubMed1992

Perceptual organization and attention.

It is widely assumed that the grouping of the visual field first described by the Gestalt psychologists and the related phenomenon of texture segregation occur very early in the processing of visual information and involve preattentive processes. All the recent evidence supporting this assumption comes from visual search experiments in which the subject is actively looking for a target and attending to the stimulus. The question at issue is whether these kinds of patterns are perceived under conditions of inattention, i.e., when observers are not searching for them. We performed six experiments to determine whether texture segregation and grouping by similarity or proximity are perceived under conditions of inattention. On the first two trials subjects were asked to report the longer arm of a briefly presented cross which was surrounded by a pattern of ungrouped small elements. On the third trial and subsequent control trials these elements were configured into grouping patterns and subjects queried about them immediately following their line length reports. The results establish that neither texture segregation nor grouping by similarity of lightness or proximity are perceived under conditions of inattention. They support the conclusion that there is an earlier stage of processing than that referred to as preattentive.

Adult↗
PubMed1988

daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex.

daughterless (da) has multiple functions in Drosophila embryonic development: maternal da activity is necessary for proper sex determination, and zygotic da activity is necessary for formation of the peripheral nervous system. We have cloned the region containing da and have found that five recessive lethal da mutations map to a single transcription unit. A predicted protein product of this transcription unit has sequence similarities with the oncogene myc, with the gene MyoD1, which is involved in myoblast determination, and with the Drosophila achaete-scute complex, which is involved in neuronal precursor determination. The role of da as a gene controlling cell determination in multiple developmental pathways is discussed.

Amino Acid Sequence↗
PubMed1985

Radioprotection of the kidney with degradable microspheres. A pilot study in the dog with repeated irradiation.

The left kidney of 9 dogs was irradiated daily for three days with 300 kVp X-rays. Radioprotection was attempted in 5 of the kidneys by administration of degradable microspheres in the renal artery, immediately before irradiation. Protected kidneys received 3 X 8 or 3 X 12 Gy in midplane dose, unprotected kidneys 3 X 5.2 or 3 X 8 Gy. A protective effect due to induced hypoxia could be demonstrated in creatinine clearance, which was significantly higher in protected than in unprotected kidneys at 7 - 13 weeks after irradiation.

Animals↗
PubMed1985

Origin and time course of gas bubbles following rapid decompression in the hamster.

Because conflicting results have been obtained in studies of the hamster (Mesocricetus auratus) microcirculation following rapid decompression, we were interested in the origin of bubbles in the cheek-pouch preparation. The hamster cheek-pouch and surgically exposed femoral vessels were observed under the microscope, before and immediately following rapid decompression. The animals were placed in a hyperbaric chamber and exposed to an increased pressure of 7 ATA for 1 h. They were then decompressed at a rate of 18 m/min until reaching 1 ATA. This hyperbaric exposure produced an LD50 in 100 hamsters examined in this study. In 40% of these animals "bubbles" were seen entering the cheek-pouch artery after a delay of from 3 to 6 min following decompression. Before observing bubbles in the cheek-pouch artery, bubbles were always seen in the femoral vein. Those animals that had observable bubbles in the cheek-pouch usually demonstrated a tachypnea, followed by gasping and apnea. When bubbles were seen in the arterial circulation, few animals survived for more than 10 min following the hyperbaric exposure. Postmortem examination of these animals revealed massive gas emboli in the large venous vessels, right ventricle, and fewer bubbles in the left ventricle. Postmortem examination of surviving animals revealed that the majority of the gas was in the venous vessels and right heart. These data support the hypothesis that bubbles first form on the venous side of the circulation and, if they exceed a certain volume, move through the pulmonary circulation into the systemic arterial vessels.

Animals↗