Academic Appointments

  • Basic Life Science Research Associate, Biology

Honors & Awards

  • Postdoctoral fellowship, Hereditary Disease Foundation

Boards, Advisory Committees, Professional Organizations

  • Chair of the organizing committee, Biophysical Society Thematic Meeting: "Revisiting the Central Dogma at the Single Molecule Level" (2018 - 2019)

All Publications

  • A Practical Guide to Graduate School Interviewing for Historically Excluded Individuals. American journal of physiology. Heart and circulatory physiology Ransey, E., Brookens, S., Beasley, H. K., Marshall, A., Marlin, B. J., Rodriguez-Aliaga, P., Headley, C. A., Wanjalla, C., Vazquez, A. D., Murray, S., Damo, S., Taabazuing, C. Y., Hinton, A. J. 2023

    View details for DOI 10.1152/ajpheart.00123.2023

    View details for PubMedID 37027327

  • Dissecting the structural basis of Huntingtin pathogenesis: one molecule at the time Rodriguez-Aliaga, P., Sosa, R. P., Bustamante, C., Frydman, J. CELL PRESS. 2022: 22
  • Knots can impair protein degradation by ATP-dependent proteases. Proceedings of the National Academy of Sciences of the United States of America San Martín, Á. n., Rodriguez-Aliaga, P. n., Molina, J. A., Martin, A. n., Bustamante, C. n., Baez, M. n. 2017; 114 (37): 9864–69


    ATP-dependent proteases translocate proteins through a narrow pore for their controlled destruction. However, how a protein substrate containing a knotted topology affects this process remains unknown. Here, we characterized the effects of the trefoil-knotted protein MJ0366 from Methanocaldococcus jannaschii on the operation of the ClpXP protease from Escherichia coli ClpXP completely degrades MJ0366 when pulling from the C-terminal ssrA-tag. However, when a GFP moiety is appended to the N terminus of MJ0366, ClpXP releases intact GFP with a 47-residue tail. The extended length of this tail suggests that ClpXP tightens the trefoil knot against GFP, which prevents GFP unfolding. Interestingly, if the linker between the knot core of MJ0366 and GFP is longer than 36 residues, ClpXP tightens and translocates the knot before it reaches GFP, enabling the complete unfolding and degradation of the substrate. These observations suggest that a knot-induced stall during degradation of multidomain proteins by AAA proteases may constitute a novel mechanism to produce partially degraded products with potentially new functions.

    View details for DOI 10.1073/pnas.1705916114

    View details for PubMedID 28847957

    View details for PubMedCentralID PMC5604015

  • Substrate-translocating loops regulate mechanochemical coupling and power production in AAA plus protease C1pXP NATURE STRUCTURAL & MOLECULAR BIOLOGY Rodriguez-Aliaga, P., Ramirez, L., Kim, F., Bustamante, C., Martin, A. 2016; 23 (11): 974-981


    ATP-dependent proteases of the AAA+ family, including Escherichia coli ClpXP and the eukaryotic proteasome, contribute to maintenance of cellular proteostasis. ClpXP unfolds and translocates substrates into an internal degradation chamber, using cycles of alternating dwell and burst phases. The ClpX motor performs chemical transformations during the dwell and translocates the substrate in increments of 1-4 nm during the burst, but the processes occurring during these phases remain unknown. Here we characterized the complete mechanochemical cycle of ClpXP, showing that ADP release and ATP binding occur nonsequentially during the dwell, whereas ATP hydrolysis and phosphate release occur during the burst. The highly conserved translocating loops within the ClpX pore are optimized to maximize motor power generation, the coupling between chemical and mechanical tasks, and the efficiency of protein processing. Conformational resetting of these loops between consecutive bursts appears to determine ADP release from individual ATPase subunits and the overall duration of the motor's cycle.

    View details for DOI 10.1038/nsmb.3298

    View details for Web of Science ID 000386992700007

    View details for PubMedID 27669037

  • New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase–promoter complex Nucleic Acids Research Doniselli*, N., Rodriguez-Aliaga*, P., Amidani, D., Bardales, J. A., Bustamante, C., Guerra, D. G., Rivetti, C. 2015; 43 (10): 5249–5262


    The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

    View details for DOI 10.1093/nar/gkv391

    View details for PubMedCentralID PMC4446441

  • Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases. Nanoscale Cheng, B., Wu, S., Liu, S., Rodriguez-Aliaga, P., Yu, J., Cui, S. 2015; 7 (7): 2970-7


    Most proteins are typically folded into predetermined three-dimensional structures in the aqueous cellular environment. However, proteins can be exposed to a nonpolar environment under certain conditions, such as inside the central cavity of chaperones and unfoldases during protein degradation. It remains unclear how folded proteins behave when moved from an aqueous solvent to a nonpolar one. Here, we employed single-molecule atomic force microscopy and molecular dynamics (MD) simulations to investigate the structural and mechanical variations of a polyprotein, I278, during the change from a polar to a nonpolar environment. We found that the polyprotein was unfolded into an unstructured polypeptide spontaneously when pulled into nonpolar solvents. This finding was corroborated by MD simulations where I27 was dragged from water into a nonpolar solvent, revealing details of the unfolding process at the water/nonpolar solvent interface. These results highlight the importance of water in maintaining folding stability, and provide insights into the response of folded proteins to local hydrophobic environments.

    View details for DOI 10.1039/c4nr07140a

    View details for PubMedID 25597693

  • The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears CELL Sen, M., Maillard, R. A., Nyquist, K., Rodriguez-Aliaga, P., Presse, S., Martin, A., Bustamante, C. 2013; 155 (3): 636-646


    ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force-generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant "rpm" but uses different "gears."

    View details for DOI 10.1016/j.cell.2013.09.022

    View details for Web of Science ID 000326571800017

    View details for PubMedID 24243020