Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering

Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I-1, I-2, and I-3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I-1 intermediate is generated within 100 ps and transforms to the R-like I-2 intermediate with a time constant of 3.2 +/- 0.2 ns. Subsequently, the late, T-like I-3 intermediate is formed via subunit rotation, a decrease in the heme-heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 +/- 120 ns for the fully photolyzed form and 5.6 +/- 0.8 mu s for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I-3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme-heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.1149sciescopu

Complete Chloroplast Genomes of Erianthus arundinaceus and Miscanthus sinensis: Comparative Genomics and Evolution of the Saccharum Complex.

The genera Erianthus and Miscanthus, both members of the Saccharum complex, are of interest as potential resources for sugarcane improvement and as bioenergy crops. Recent studies have mainly focused on the conservation and use of wild accessions of these genera as breeding materials. However, the sequence data are limited, which hampers the studies of phylogenetic relationships, population structure, and evolution of these grasses. Here, we determined the complete chloroplast genome sequences of Erianthus arundinaceus and Miscanthus sinensis by using 454 GS FLX pyrosequencing and Sanger sequencing. Alignment of the E. arundinaceus and M. sinensis chloroplast genome sequences with the known sequence of Saccharum officinarum demonstrated a high degree of conservation in gene content and order. Using the data sets of 76 chloroplast protein-coding genes, we performed phylogenetic analysis in 40 taxa including E. arundinaceus and M. sinensis. Our results show that S. officinarum is more closely related to M. sinensis than to E. arundinaceus. We estimated that E. arundinaceus diverged from the subtribe Sorghinae before the divergence of Sorghum bicolor and the common ancestor of S. officinarum and M. sinensis. This is the first report of the phylogenetic and evolutionary relationships inferred from maternally inherited variation in the Saccharum complex. Our study provides an important framework for understanding the phylogenetic relatedness of the economically important genera Erianthus, Miscanthus, and Saccharum

Microsatellites in <i>Erianthus arundinaceus</i> and <i>Miscanthus sinensis</i> chloroplast genomes.

<p>Microsatellites in <i>Erianthus arundinaceus</i> and <i>Miscanthus sinensis</i> chloroplast genomes.</p

Alignment of whole chloroplast genome sequences from four Panicoideae species.

<p>Chloroplast genomes were aligned by using the mVISTA program with the <i>Zea mays</i> sequence as a reference. The <i>X</i>- and <i>Y</i>-scales indicate the coordinates within cp genomes and the percentage of identity (50%–100%), respectively. Genome regions (exons, introns, and conserved non-coding sequences) are color-coded. Gray arrows indicate the direction of transcription of each gene. The genes encoding transfer RNAs (<i>trn</i>) are indicated under gray arrows using the single-letter amino acid code (e.g., K: <i>trn</i>K).</p

Divergence times of the PACMAD clade.

<p>A Bayesian relaxed-clock approach based on 76 concatenated protein-coding chloroplast genes was used to estimate divergence times.</p

Phylogenetic analysis of 40 species including three genera of the <i>Saccharum</i> complex.

<p>A phylogenetic tree was generated using the maximum-likelihood method based on the concatenated nucleotide sequences of 76 protein-coding chloroplast genes. Numbers beside the nodes indicate the bootstrap values (%) from 1,000 replicates.</p