Binding of the hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to synthetic polypeptides and proteins with a different structural organization has been studied. It has been shown that ANS has a much stronger affinity to the protein "molten globule" state, with a pronounced secondary structure and compactness, but without a tightly packed tertiary structure as compared with its affinity to the native and coil-like proteins, or to coil-like, alpha-helical, or beta-structural hydrophilic homopolypeptides. The possibility of using ANS for the study of equilibrium and kinetic molten globule intermediates is demonstrated, with carbonic anhydrase, beta-lactamase, and alpha-lactalbumin as examples.
The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.
We report the fast relaxation dynamics of ''native'' apomyoglobin (pH 5.3) following a 10-ns, laserinduced temperature jump. The structural dynamics are probed using time-resolved infrared spectroscopy. The infrared kinetics monitored within the amide I absorbance of the polypeptide backbone exhibit two distinct relaxation phases which have different spectral signatures and occur on very different time scales ( ؍ 1633 cm ؊1 , ؍ 48 ns; ؍ 1650 cm ؊1 , ؍ 132 s). We assign these two spectral components to discrete substructures in the protein: helical structure that is solvated (1633 cm ؊1 ) and native helix that is protected from solvation by interhelix tertiary interactions (1650 cm ؊1 ). Folding rate coefficients inferred from the observed relaxations at 60؇C are k f(solvated) ؍ (7 to 20) ؋ 10 6 s ؊1 and k f(native) ؍ 3.6 ؋ 10 3 s ؊1 , respectively. The faster rate is interpreted as the intrinsic rate of solvated helix formation, whereas the slower rate is interpreted as the rate of formation of tertiary contacts that determine a native helix. Thus, at 60؇C helix formation precedes the formation of tertiary structure by over three orders of magnitude in this protein. Furthermore, the distinct thermodynamics and kinetics observed for the apomyoglobin substructures suggest that they fold independently, or quasi-independently. The observation of inhomogeneous folding for apomyoglobin is remarkable, given the relatively small size and structural simplicity of this protein.The mechanisms by which a protein searches vast conformational space to attain its native fold in reasonable times and by which the three-dimensional structure is encoded in the primary sequence have not been resolved experimentally. In particular, the critical early-time structural dynamics which carry a protein along the pathway(s) from extended, disordered conformations to a compact fold are poorly characterized. A major impediment has been the conventional solutionmixing approach to initiation of a folding reaction, which imposes a short-time observation limit of greater than 1 ms.
We have developed a rapid molecular mapping technology-Direct Linear Analysis (DLA)-on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of ±0.8 kb, and these tags could be localized on the DNA with an accuracy of ±2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules.[Supplemental material is available online at www.genome.org.]Traditionally, DNA mapping has been an important strategy to study structures and organizations of genomes. Recent advances in DNA sequencing technologies, however, have served to diminish the relative importance of traditional mapping. Nonetheless, growing interest in comparative genomics has created a need for technologies that can rapidly and efficiently characterize a genome, particularly larger genomes. Furthermore, in many cases, single-base resolution is unnecessary, as genomic differences among species (e.g., microorganisms) or among individuals within a given species (e.g., humans) can be discerned using lower-resolution mapping approaches (Olive and Bean 1999). Thus, there is a need for a practical, rapid, and highly efficient DNA mapping technology.Currently, restriction mapping is the most practicable mapping approach that combines high resolution with high density (Brown 1999). Gel electrophoresis-based restriction enzyme mapping using just a single enzyme has been a workhorse for the human genome project and other large-scale efforts to provide a fingerprint identification of BAC clones (Soderlund et al. 2000). Traditional restriction mapping with multiple enzymes has allowed characterization and manipulation of genomic regions of interest (Brown 1999). To study human variation, restriction fragment length polymorphism (RFLP) analysis has allowed investigators to identify SNPs that correlate with disease loci (Shi 2002). Nonetheless, restriction mapping has fundamental drawbacks that limit its utility for comparative genomics. Digestion of the DNA removes information regarding the ordering of the fragments, requiring the use of multiple enzymes to construct the correct map. Furthermore, as RFLP analysis involves a mixture of molecules, haplotype information is inaccessible. For large DNA, pulsed-fi...
R. Brian Dyer received the Ph.D. degree in inorganic chemistry from Duke University in 1985. After spending 2 years in the Inorganic and Structural Chemistry Group (INC-4) at Los Alamos National Laboratory as a postdoc, he moved to the chemical and laser sciences division as a staff member. He is currently on staff in the biosciences and biotechnology group (CST-4). He was recently awarded the Los Alamos Fellows Prize for his work on molecular dynamics and protein folding.
Most experimental studies on the dynamics of protein folding have been confined to timescales of 1 ms and longer. Yet it is obvious that many phenomena that are obligatory elements of the folding process occur on much faster timescales. For example, it is also now clear that the formation of secondary and tertiary structures can occur on nanosecond and microsecond times, respectively. Although fast events are essential to, and sometimes dominate, the overall folding process, with a few exceptions their experimental study has become possible only recently with the development of appropriate techniques. This review discusses new approaches that are capable of initiating and monitoring the fast events in protein folding with temporal resolution down to picoseconds. The first important results from those techniques, which have been obtained for the folding of some globular proteins and polypeptide models, are also discussed.
High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.
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