Stretching single-domain proteins: Phase diagram and kinetics of force-induced unfolding

Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a stepwise “quantized” manner. Unfolding dynamics and forces required to stretch proteins depend sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value, which is determined by the barrier to unfolding when f = 0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single-molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.

Using O2 to probe membrane immersion depth by 19F NMR

A fluorinated detergent, CF3(CF2)5C2H4-O-maltose, was reconstituted into a lipid bilayer model membrane system to demonstrate the feasibility of determining solvent accessibility and membrane immersion depth of each fluorinated group by 19F NMR. Apolar oxygen, which is known to partition with an increasing concentration gradient toward the hydrophobic membrane interior, exhibits a range of paramagnetic relaxation effects on 19F nuclei, depending on its depth in the membrane. This effect, which is predominately associated with spin-lattice relaxation rates (R1) and chemical shifts, can be amplified greatly with minimal line broadening by increasing the partial pressure of O2 at least 100-fold (i.e., PO2 greater than 20 bar). The differences of longitudinal relaxation rates at 20 bar of oxygen pressure to those under ambient pressure (R120bar − R10) are largest for those fluorine groups expected to be most deeply buried in the membrane bilayer. This result contrasts with the reverse trend, which is observed on addition of a membrane surface-associated paramagnetic species, 4-(N,N-dimethyl-N-hexadecyl) ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16) at ambient pressures. Thus, differential relaxation rates may be observed in 19F-labeled membrane-associated molecules resulting from the addition of apolar oxygen under high pressure. The results demonstrate that the degree of solvent accessibility and membrane immersion depth of specific fluorinated species in membrane-associated macromolecules can be probed by 19F NMR.

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples.

Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and Synthesis of Structurally Defined Fructan Molecules from Sucrose1

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins.

Fos and Jun bend the AP-1 site: effects of probe geometry on the detection of protein-induced DNA bending.

The effect of Fos and Jun binding on the structure of the AP-1 recognition site is controversial. Results from phasing analysis and phase-sensitive detection studies of DNA bending by Fos and Jun have led to opposite conclusions. The differences between these assays, the length of the spacer between two bends and the length of the sequences flanking the bends, are investigated here using intrinsic DNA bend standards. Both an increase in the spacer length as well as a decrease in the length of flanking sequences resulted in a reduction in the phase-dependent variation in electrophoretic mobilities. Probes with a wide separation between the bends and short flanking sequences, such as those used in the phase-sensitive detection studies, displayed no phase-dependent mobility variation. This shape-dependent variation in electrophoretic mobilities was reproduced by complexes formed by truncated Fos and Jun. Results from ligase-catalyzed cyclization experiments have been interpreted to indicate the absence of DNA bending in the Fos-Jun-AP-1 complex. However, truncated Fos and Jun can alter the relative rates of inter- and intramolecular ligation through mechanisms unrelated to DNA bending, confounding the interpretation of cyclization data. The analogous phase- and shape-dependence of the electrophoretic mobilities of the Fos-Jun-AP-1 complex and an intrinsic DNA bend confirm that Fos and Jun bend DNA, which may contribute to their functions in transcription regulation.

A stationary-phase stress-response sigma factor from Mycobacterium tuberculosis.

Alternative RNA polymerase sigma factors are a common means of coordinating gene regulation in bacteria. Using PCR amplification with degenerate primers, we identified and cloned a sigma factor gene, sigF, from Mycobacterium tuberculosis. The deduced protein encoded by sigF shows significant similarity to SigF sporulation sigma factors from Streptomyces coelicolor and Bacillus subtilis and to SigB, a stress-response sigma factor, from B. subtilis. Southern blot surveys with a sigF-specific probe identified cross-hybridizing bands in other slow-growing mycobacteria, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium avium, but not in the rapid-growers Mycobacterium smegmatis or Mycobacterium abscessus. RNase protection assays revealed that M. tuberculosis sigF mRNA is not present during exponential-phase growth in M. bovis BCG cultures but is strongly induced during stationary phase, nitrogen depletion, and cold shock. Weak expression of M. tuberculosis sigF was also detected during late-exponential phase, oxidative stress, anaerobiasis, and alcohol shock. The specific expression of M. tuberculosis sigF during stress or stationary phase suggests that it may play a role in the ability of tubercle bacilli to adapt to host defenses and persist during human infection.

Liquid-phase combinatorial synthesis.

A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of nonpeptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. & Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against beta-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained.

Estudo da microestrutura e dinâmica molecular do Poly(3-(2\'-ethylhexyl)thiophene)(P3EHT) via ressonância magnética nuclear

O estudo da microestrutura e dinâmica molecular de polímeros conjugados é de grande importância para o entendimento das propriedades físicas desta classe de materiais. No presente trabalho utilizou-se técnicas de ressonância magnética nuclear em baixo e alto campo para elucidar os processos de dinâmica molecular e cristalização do polímero Poly(3-(2’-ethylhexyl)thiophene) - P3EHT. O P3EHT é um polímero modelo para tal estudo, pois apresenta temperatura de fusão bem inferior a sua temperatura de degradação. Esta característica permite acompanhar os processos de cristalização in situ utilizando RMN. Além disso, sua similaridade ao já popular P3HT o torna um importante candidato a camada ativa em dispositivos eletrônicos orgânicos. O completo assinalamento do espectro de 13C para o P3EHT foi realizado utilizando as técnicas de defasamento dipolar e HETCOR. Os processos de dinâmica molecular, por sua vez, foram sondados utilizando DIPSHIFT. Observou-se um gradiente de mobilidade na cadeia lateral do polímero. Além disso, os baixos valores de parametros de ordem obtidos em comparação a experimentos similares realizados no P3HT na literatura indicam um aparente aumento no volume livre entre cadeias consecutivas na fase cristalina. Isso indica que a presença do grupo etil adicional no P3EHT causa um completo rearranjo das moléculas e dificulta seu empacotamento. Constatou-se ainda pouca variação das curvas de DIPSHIFT para os carbonos da cadeia lateral como função do método de excitação utilizado, o que aponta para um polímero que apresenta cadeia lateral móvel mesmo em sua fase cristalina. Os dados de dinâmica molecular foram corroborados por medidas de T1, T1ρ e TCH. Utilizando filtros dipolares em baixo campo observou-se três temperaturas de transição para o P3EHT: 250 K, 325 K e 350 K. A cristalização desse material é um processo lento. Verificou-se que o mesmo pode se estender por até até 24h a temperatura ambiente. Mudanças no espectro de 13C utilizando CPMAS em alto campo indicam um ordenamento dos anéis tiofeno (empacotamento π – π) como o principal processo de cristalização para o P3EHT.

Spin dynamics of current-driven single magnetic adatoms and molecules

A scanning tunneling microscope can probe the inelastic spin excitations of a single magnetic atom in a surface via spin-flip assisted tunneling in which transport electrons exchange spin and energy with the atomic spin. If the inelastic transport time, defined as the average time elapsed between two inelastic spin flip events, is shorter than the atom spin-relaxation time, the scanning tunnel microscope (STM) current can drive the spin out of equilibrium. Here we model this process using rate equations and a model Hamiltonian that describes successfully spin-flip-assisted tunneling experiments, including a single Mn atom, a Mn dimer, and Fe Phthalocyanine molecules. When the STM current is not spin polarized, the nonequilibrium spin dynamics of the magnetic atom results in nonmonotonic dI/dV curves. In the case of spin-polarized STM current, the spin orientation of the magnetic atom can be controlled parallel or antiparallel to the magnetic moment of the tip. Thus, spin-polarized STM tips can be used both to probe and to control the magnetic moment of a single atom.

Local probe of fractional edge states of S=1 Heisenberg spin chains

Spin chains are among the simplest physical systems in which electron-electron interactions induce novel states of matter. Here we propose to combine atomic scale engineering and spectroscopic capabilities of state of the art scanning tunnel microscopy to probe the fractionalized edge states of individual atomic scale S=1 spin chains. These edge states arise from the topological order of the ground state in the Haldane phase. We also show that the Haldane gap and the spin-spin correlation length can be measured with the same technique.

Experimental study of phase equilibria in the Fe-Sn-Zn-O system in air

The four-component Fe-Sn-Zn-O system was studied experimentally in the range of temperatures from 1100 to 1400 degrees C in air using high temperature equilibration and quenching techniques followed by electron probe X-ray microanalysis (EPMA). Phase equilibrium relations and the extent of solid solutions among the phases cassiterite (Sn,Zn)O-2, hematite (Fe,Sn,Zn)(2)O-3, spinel (Fe,Sn,Zn)(3)O-4 and zincite (Zn,Fe,Sn)O are reported. Phase equilibria in the pseudo-binary systems Fe2O3-SnO2 and SnO2-ZnO are reported in air in the temperature ranges from 1100 to 1400 degrees C and 1200 to 1400 degrees C, respectively.

Experimental study of phase equilibria in the Al-Fe-Zn-O system in air

The phase equilibria in the Al-Fe-Zn-O system in the range 1250 °C to 1695 °C in air have been experimentally studied using equilibration and quenching techniques followed by electron probe X-ray microanalysis. The phase diagram of the binary Al2O3-ZnO system and isothermal sections of the Al2O3-“Fe2O3”-ZnO system at 1250 °C, 1400 °C, and 1550 °C have been constructed and reported for the first time. The extents of solid solutions in the corundum (Al,Fe)2O3, hematite (Fe,Al)2O3, Al2O3*Fe2O3 phase (Al,Fe)2O3, spinel (Al,Fe,Zn)O4, and zincite (Al,Zn,Fe)O primary phase fields have been measured. Corundum, hematite, and Al2O3*Fe2O3 phases dissolve less than 1 mol pct zinc oxide. The limiting compositions of Al2O3*Fe2O3 phase measured in this study at 1400 °C are slightly nonstoichiometric, containing more Al2O3 then previously reported. Spinel forms an extensive solid solution in the Al2O3-“Fe2O3”-ZnO system in air with increasing temperature. Zincite was found to dissolve up to 7 mole pct of aluminum in the presence of iron at 1550 °C in air. A meta-stable Al2O3-rich phase of the approximate composition Al8FeZnO14+x was observed at all of the conditions investigated. Aluminum dissolved in the zincite in the presence of iron appears to suppress the transformation from a round to platelike morphology.

Phase equilibria at sub-solidus conditions in the Fe-Mg-Zn-O system in air

The phase equilibria in the Fe-Mg-Zn-O system in the temperature range 1100-1550degreesC in air have been experimentally studied using equilibration and quenching followed by electron probe X-ray microanalysis. The compositions of condensed phases in equilibrium in the binary MgO-ZnO system and the ternary Fe-Mg-O system have been reported at sub-solidus in air. Pseudo-ternary sections of the quaternary Fe-Mg-Zn-O system at 1100, 1250 and 1400degreesC in air were constructed using the experimental data. The solid solution of iron oxide, MgO and ZnO in the periclase (Mg, Zn, Fe)O, spinel (Mg2+, Fe2+, Zn2+)(x)Fe(2+y)3+O4 and zincite (Zn, Mg, Fe)O phases were found to be extensive under the conditions investigated. A continuous spinel solid solution is formed between the magnesioferrite (Mg2+, Fe2+)(x)Fe(2+y)3+O4 and franklinite (Zn2+, Fe2+)(x)Fe(2+y)3+O4 end-members at 1100 and 1250degreesC, extending to magnetite (Fe2+)(x)Fe(2+y)3+O4 at 1400degreesC in air. The compositions along the spinel boundaries were found to be non-stoichiometric, the magnitude of the non-stoichiometry being a function of composition and temperature in air. It was found that hematite dissolves neither MgO nor ZnO in air.