1. Volume 26, Issue 3, Pages 426-436.e3 (March 2018)
Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily 
Chitra Narayanan, David N. Bernard, Khushboo Bafna, Donald Gagné, Chakra S. Chennubhotla, Nicolas Doucet, Pratul K. Agarwal 
Structure 
Volume 26, Issue 3, Pages e3 (March 2018)
DOI: /j.str
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2. Structure 2018 26, 426-436.e3DOI: (10.1016/j.str.2018.01.015)
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3. Figure 1 Structural and Phylogenetic Analysis of RNase Homologs
(A) Consensus sequence alignment of the 23 RNase homologs, with the corresponding sequence identifiers displayed to the left of each sequence. Detailed information about the sequences is provided in Table S1. The secondary structural elements α helices (orange rectangles), β strands (cyan arrows), and loop regions (L), corresponding to the bovine RNase A structure, are displayed above the alignment. Conserved active-site residues His12, Lys41, and His119 (BtRA numbering) are identified with an asterisk above the alignment. BtRA (PDB: 7RSA) sequence numbering (BtRA#) is shown at the top and consensus sequence numbering (Cons#) below the multiple sequence alignment. A list of all consensus sequence positions is presented in Figure S2. Gaps in the alignment are represented using dotted lines.
(B) Structural alignment of representative RNase homologs. Structures include 7RSA (green), 1QMT (blue), 2HKY (cyan), 1ANG (salmon), 3SNF (yellow), and 1RNF (gold). The V1 and V2 arms are identified by rectangular boxes. Active-site residues are displayed as sticks and labeled with BtRA numbering.
(C) Phylogenetic clustering of the 23 RNases generated based on maximum-likelihood analysis. Sequences are classified into four clusters, identified using different colors. Alignments were prepared using EsPript (Robert and Gouet, 2014).
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4. Figure 2
Millisecond Conformational Exchange Experienced by Select Hominidae and Bovine RNases
Cartoon representation of: (A) EAR-like subfamily members: Homo sapiens EDN (HsR2), H. sapiens ECP (HsR3), Pongo pygmaeus ECP (PpR3), Macaca fascicularis ECP (MfR3); (B) RNase A-like subfamily member Bos taurus RNase A (BtRA) and the BtRAHsR3 chimera (Doucet et al., 2009); (C) Angiogenin-like subfamily member H. sapiens angiogenin (HsR5); and (D) H. sapiens RNase 4 (HsR4) from the Others cluster. Structures are colored based on the phylogenetic group shown in Figure 1C. Residues identified by NMR as undergoing conformational exchange on the millisecond timescale are depicted as spheres, and numbered according to their position in the sequence alignment (see Figure 1) to facilitate comparison. Conformational exchange was probed by 15N-CPMG NMR relaxation dispersion experiments at 500 and 800 MHz (298 K). Residues were considered for further analysis only if the difference in measured R2 (1/τcp) values at fast (τcp = 0.625 ms) and slow (τcp = 10 ms) refocusing pulse delays was greater than 2 s−1, similar to previous reports (Doucet et al., 2009; Gagné et al., 2012). BtRAHsR3, which displays subsector conformational exchange similar to HsR3, is an artificial chimeric hybrid of BtRA in which the 12-residue long loop 1 of BtRA (14-DSSTSAASSSNY-25, BtRA numbering) was replaced by the 6-residue long loop 1 of HsR3 (17-SLNPPR-22, HsR3 numbering) (Gagné et. al., 2012). Structures color-coded based on conformational exchange rates are shown in Figure S4.
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5. Figure 3 Dynamical Effects of Loop 1 Swapping on BtRA
(A) Relaxation dispersion profiles of various loop 1 residues obtained from 15N-CPMG experiments at 800 MHz and 25°C for bovine RNase A (BtRA), human RNase 3 (HsR3), and a previously described (Doucet et al., 2009) chimera of RNase A (BtRAHsR3) in which loop 1 has been replaced by the corresponding residues of HsR3. 15N-CPMG data was also acquired at 500 and/or 600 MHz for these proteins (not shown for clarity). Solid curves are shown for residues displaying conformational exchange, and dashed curves belong to non-exchanging residues. Curves of the same color represent structurally equivalent residues in the sequence alignment.
(B) Compounded 1H-15N chemical-shift variations (Grzesiek et al., 1996) induced on BtRA by loop 1 swapping in BtRAHsR3. Residues in gray are either unassigned or belong to the swapped loop. Chemical-shift variations for each residue are indicated by both the color and the width of the cartoon putty. Figure was made with PyMOL using the bovine RNase A structure (PDB: 7RSA).
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6. Figure 4 Microsecond Conformational Dynamics of RNase Homologs
Root-mean-square fluctuations corresponding to the Cα displacements in the top ten quasi-harmonic modes (RMSF10), for the 23 proteins grouped into the four phylogenetic clusters described in Figure 1. A consensus sequence with gaps is used (see Figure 1A), where gaps are represented as dotted regions for each protein. The panel on the right shows representative RNase homologs from each phylogenetic group using a tube representation where the thickness of the tube corresponds to the flexibility of residues in each protein, with thicker tubes corresponding to flexible regions and thinner tubes representing less flexible regions. The dynamical range represented using the color spectrum is consistent across all sequences with the blue and red ends of the spectrum corresponding to low and high dynamic regions, respectively. The representative structures correspond to B. taurus RNase A (BtRA) and H. sapiens RNase 1 (HsR1); H. sapiens RNase 3 (HsR3) and P. pygmaeus RNase 3 (PpR3); H. sapiens angiogenin (HsR5) and Mus musculus RNase 5 (MmR5); and H. sapiens RNase 4 (HsR4) and Rana pipiens RNase (RpRx), respectively. N-terminal residues, which showed larger RMSFs (see left panel), are hidden for clarity. Tube representations for all RNases are shown in Figure S5.
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7. Figure 5 Dynamical Properties of BtRAHsR3 Chimera
(A) Comparison of the root-mean-square fluctuations of the ten slowest modes (RMSF10) between BtRA (black) and the chimera (red). On the right, RMSFs are shown using a tube representation for BtRA and the chimera, where the thickness of the tube corresponds to the flexibility of residues in each protein, with thicker tubes corresponding to flexible regions and thinner tubes representing less flexible regions.
(B) Dynamical cross-correlation maps for BtRA (left), with regions displaying significant correlations and anti-correlations identified using boxes, and the BtRAHsR3 chimera (right). Loops 1, 2, 4, and 6 are highlighted in yellow.
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8. Figure 6 Quantitative Characterization of Dynamical Similarities
Pairwise Pearson's correlations for the 23 RNase homologs classified into four phylogenetic clusters.
(A) Correlation coefficients for each pair of sequences, color-coded based on strong correlations (red) to weaker or no correlations (gray).
(B–D) Average correlations, calculated by averaging the correlation coefficients for sequences compared for: all residue positions (B), consensus positions (C), and non-consensus positions, corresponding to insertions or deletions in sequences (D). The diagonal elements correspond to correlations within phylogenetic subfamilies while off-diagonal elements correspond to correlations between the subfamilies. Standard deviations (SD) for average correlation calculations are shown in parentheses. Abbreviations R, E, A, and O correspond to phylogenetic clusters RNase A-like, EAR-like, Angiogenin-like, and Others, respectively.
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