The G-rich strand of human telomeric DNA can fold into a four-stranded structure called G-quadruplex and inhibit telomerase activity that is expressed in 85-90% tumor cells. For this reason, telomere quadruplex is emerging as a potential therapeutic target for cancer. Information on the structure of the quadruplex in the physiological environment is important for structure-based drug design targeting the quadruplex. Recent studies have raised significant controversy regarding the exact structure of the quadruplex formed by human telomeric DNA in a physiological relevant environment. Studies on the crystal prepared in K+ solution revealed a distinct propeller-shaped parallel-stranded conformation. However, many later works failed to confirm such structure in physiological K+ solution but rather led to the identification of a different hybrid-type mixed parallel/antiparallel quadruplex. Here we demonstrate that human telomere DNA adopts a parallel-stranded conformation in physiological K+ solution under molecular crowding conditions created by PEG. At the concentration of 40% (w/v), PEG induced complete structural conversion to a parallel-stranded G-quadruplex. We also show that the quadruplex formed under such a condition has unusual stability and significant negative impact on telomerase processivity. Since the environment inside cells is molecularly crowded, our results obtained under the cell mimicking condition suggest that the parallel-stranded quadruplex may be the more favored structure under physiological conditions, and drug design targeting the human telomeric quadruplex should take this into consideration.

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on nativelike interfoldon interactions orders the pathway.D o proteins fold through varied and multiple tracks, or do they fold through predetermined intermediates according to understandable biophysical principles (1)? This question is fundamental for the interpretation of a large amount of biophysical and biological research. The question could be resolved if it were possible to define the intermediate structures and pathways that unfolded proteins move through on their way to the native state. Unfortunately, transient intermediates cannot be studied by the usual crystallographic and NMR methods. The range of kinetic and spectroscopic methods has been applied to many proteins, but these methods do not yield the necessary structural information.We used a developing technology, hydrogen exchange pulse labeling measured by MS (HX MS), to study the folding of a cysteine-free variant of Escherichia coli ribonuclease H1 (RNase H), a mixed α/β protein that has served as a major proteinfolding model (2-5). Previous studies showed that RNase H folds in a fast, unresolved burst phase (15 ms dead time) to an intermediate termed "I core " and then much more slowly (in seconds) to the native state (3). HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2-4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6, 7).The HX MS technique used here is able to follow the entire folding trajectory of RNase H in considerable structural and temporal detail. The analysis monitors every amide site, evaluates the folding coopera...

Hydrogen exchange technology provides a uniquely powerful instrument for measuring protein structural and biophysical properties, quantitatively and in a nonperturbing way, and determining how these properties are implemented to produce protein function. A developing hydrogen exchange-mass spectrometry method (HX MS) is able to analyze large biologically important protein systems while requiring only minuscule amounts of experimental material. The major remaining deficiency of the HX MS method is the inability to deconvolve HX results to individual amino acid residue resolution. To pursue this goal we used an iterative optimization program (HDsite) that integrates recent progress in multiple peptide acquisition together with previously unexamined isotopic envelope-shape information and a site-resolved backexchange correction. To test this approach, residue-resolved HX rates computed from HX MS data were compared with extensive HX NMR measurements, and analogous comparisons were made in simulation trials. These tests found excellent agreement and revealed the important computational determinants.HDX-MS | isotope pattern | protein biophysics U nlike any other method, hydrogen exchange (HX) behavior encodes detailed quantitative information at amino acid resolution on the biophysical factors that produce protein function-structure, structure change, interactions, dynamics, and energetics. HX behavior is very sensitive to these properties, and the chemistry (1, 2) and structural physics (3, 4) of HX processes in these terms are now well understood. The ability of HX to measure protein biophysical properties and their implementation in protein function has been demonstrated over the last 30 y by many NMR studies. However, routine NMR analysis is limited to small highly soluble proteins that can be obtained in quantity (multi mgs), isotopically labeled ( 15 N, 13 C), and studied at millimolar concentration. A developing technology, HX measured and analyzed by mass spectrometry (HX MS) (5-16) can extend this proven capability to the much larger and more complex protein systems that make biology work. The method requires only picomoles of protein at submicromolar concentrations; it can be used to study the properties and functioning of proteins at any condition one chooses, and the experimental protein need not even be very pure.For HX MS analysis, a protein sample taken from any H-D exchange experiment is quenched into slow HX conditions, proteolytically fragmented, and the peptide fragments are separated and analyzed by HPLC and mass spectrometry. The number of D atoms carried on each peptide fragment is given by its measurable increase in mass, usually calculated as the increment in the peptide mass centroid. These results provide structural information resolved to the level of individual fragments and are broadly able to indicate where in the protein important behavior occurs (5-16). More penetrating conclusions could be drawn if it were possible to extend structural resolution to the individual amino acid level. Re...