Marianne M Lee

The Chinese University of Hong Kong, Hong Kong, Hong Kong

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Publications (11)53.38 Total impact

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    ABSTRACT: The pyrrolysine translational machinery has been extensively explored for the production of recombinant proteins containing a variety of “site-specific” non-canonical amino acids for both in vitro and in vivo biochemical studies. In this study, we report the first use of this technology for the production of branched cyclic proteins with a tadpole-like topology. As a proof of concept, we fused the well-studied RGD peptide to the C terminus of an mCherry reporter protein. Previous studies have shown that cyclization of the RGD peptide enhances its internalization into cells compared to its linear counterpart. The cellular uptake efficiencies of mCherry-cyclo(RGD), mCherry-linear(RGD), and wild-type mCherry determined by flow cytometry follow the trends expected, thereby confirming the feasibility and potential utility of this cyclization approach.
    ChemBioChem 07/2014; · 3.74 Impact Factor
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    ABSTRACT: What's the catch? A pyrrolysine analogue bearing a terminal alkyne and an ester functionality can be incorporated into recombinant proteins and render them amenable to capture by the click reaction and subsequent release through ester hydrolysis. The utility of this pyrrolysine-inspired technology is demonstrated for the identification of SUMOylation sites.
    ChemBioChem 04/2013; · 3.74 Impact Factor
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    ABSTRACT: TONs of copper fun: There is considerable interest in developing catalysts to harness the abundant natural supply of methane for various industrial applications. Two tricopper complexes capable of mediating efficient oxidation of methane to methanol under ambient conditions were tested: a biomimetic tricopper complex and a tricopper-peptide species derived from the particulate methane monooxygenase (pMMO) protein.
    Angewandte Chemie International Edition 02/2013; · 11.34 Impact Factor
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    Marianne M Lee, Michael K Chan, Ralf Bundschuh
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    ABSTRACT: A SIB-BLAST web server (http://sib-blast.osc.edu) has been established for investigators to use the SimpleIsBeautiful (SIB) algorithm for sequence-based homology detection. SIB was developed to overcome the model corruption frequently observed in the later iterations of PSI-BLAST searches. The algorithm compares resultant hits from the second iteration to the final iteration of a PSI-BLAST search, calculates the figure of merit for each 'overlapped' hit and re-ranks the hits according to their figure of merit. By validating hits generated from the last profile against hits from the first profile when the model is least corrupted, the true and false positives are better delineated, which in turn, improves the accuracy of iterative PSI-BLAST searches. Notably, this improvement to PSI-BLAST comes at minimal computational cost as SIB-BLAST utilizes existing results already produced in a PSI-BLAST search.
    Nucleic Acids Research 06/2009; 37(Web Server issue):W53-6. · 8.81 Impact Factor
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    ABSTRACT: M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted. Structural and sequence analyses implicate a highly conserved Arg at the back of the groove as being critical for this length restriction. However, the Thermus thermophilus and Bacillus subtilis M32 members lack this conserved Arg. Herein, we present the biochemical and structural characterization of these two proteins. Our findings support the important role of the conserved Arg in maintaining the length restriction, and reveal a proline-rich loop as an alternate blocking strategy. Based on our results, we propose that M32 carboxypeptidases from Bacilli belong to a separate subfamily.
    Proteins Structure Function and Bioinformatics 06/2009; 77(3):647-57. · 3.34 Impact Factor
  • Tomasz Fekner, Xin Li, Marianne M Lee, Michael K Chan
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    ABSTRACT: Ignoring the STOP sign: A pyrrolysine analogue bearing a terminal alkyne was site-specifically incorporated into recombinant calmodulin (CaM) through a UAG codon. The resulting protein was labeled with an azide-containing dye using a copper(I)-catalyzed click reaction. Subsequent application of an orthogonal cysteine tagging method yielded a CaM labeled with two distinct fluorophores that enabled its study by FRET spectroscopy.
    Angewandte Chemie International Edition 02/2009; 48(9):1633-5. · 11.34 Impact Factor
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    ABSTRACT: Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1A resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.
    Biochemical and Biophysical Research Communications 09/2008; 374(3):470-4. · 2.28 Impact Factor
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    Marianne M Lee, Michael K Chan, Ralf Bundschuh
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    ABSTRACT: MOTIVATION: The deluge of biological information from different genomic initiatives and the rapid advancement in biotechnologies have made bioinformatics tools an integral part of modern biology. Among the widely used sequence alignment tools, BLAST and PSI-BLAST are arguably the most popular. PSI-BLAST, which uses an iterative profile position specific score matrix (PSSM)-based search strategy, is more sensitive than BLAST in detecting weak homologies, thus making it suitable for remote homolog detection. Many refinements have been made to improve PSI-BLAST, and its computational efficiency and high specificity have been much touted. Nevertheless, corruption of its profile via the incorporation of false positive sequences remains a major challenge. RESULTS: We have developed a simple and elegant approach to resolve the problem of model corruption in PSI-BLAST searches. We hypothesized that combining results from the first (least-corrupted) profile with results from later (most sensitive) iterations of PSI-BLAST provides a better discriminator for true and false hits. Accordingly, we have derived a formula that utilizes the E-values from these two PSI-BLAST iterations to obtain a figure of merit for rank-ordering the hits. Our verification results based on a 'gold-standard' test set indicate that this figure of merit does indeed delineate true positives from false positives better than PSI-BLAST E-values. Perhaps what is most notable about this strategy is that it is simple and straightforward to implement.
    Bioinformatics 07/2008; 24(11):1339-43. · 5.47 Impact Factor
  • Marianne M. Lee, Michael K. Chan, Ralf Bundschuh
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    ABSTRACT: Motivation: The deluge of biological information from different genomic initiatives and the rapid advancement in biotechnologies have made bioinformatics tools an integral part of modern biology. Among the widely used sequence alignment tools, BLAST and PSI-BLAST are arguably the most popular. PSI-BLAST, which uses an iterative profile position specific score matrix (PSSM)-based search strategy, is more sensitive than BLAST in detecting weak homologies, thus making it suitable for remote homolog detection. Many refinements have been made to improve PSI-BLAST, and its computational efficiency and high specificity have been much touted. Nevertheless, corruption of its profile via the incorporation of false positive sequences remains a major challenge. Results: We have developed a simple and elegant approach to resolve the problem of model corruption in PSI-BLAST searches. We hypothesized that combining results from the first (least-corrupted) profile with results from later (most sensitive) iterations of PSI-BLAST provides a better discriminator for true and false hits. Accordingly, we have derived a formula that utilizes the E-values from these two PSI-BLAST iterations to obtain a figure of merit for rank-ordering the hits. Our verification results based on a ‘gold-standard’ test set indicate that this figure of merit does indeed delineate true positives from false positives better than PSI-BLAST E-values. Perhaps what is most notable about this strategy is that it is simple and straightforward to implement. Contact: bundschuh@mps.ohio-state.edu
    Bioinformatics. 06/2008; 24(11):1339-1343.
  • Marianne M Lee, Ralf Bundschuh, Michael K Chan
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    ABSTRACT: A new machine learning algorithm, LESTAT (LEngth and STructure-based sequence Alignment Tool) has been developed for detecting protein homologs having low-sequence identity. LESTAT is an iterative profile-based method that runs without reliance on a predefined library and incorporates several novel features that enhance its ability to identify remote sequences. To overcome the inherent bias associated with a single starting model, LESTAT utilizes three structural homologs to create a profile consisting of structurally conserved positions and block separation distances. Subsequent profiles are refined iteratively using sequence information obtained from previous cycles. Additionally, the refinement process incorporates a "lock-in" feature to retain the high-scoring sequences involved in previous alignments for subsequent model building and an enhancement factor to complement the weighting scheme used to build the position specific scoring matrix. A comparison of the performance of LESTAT against PSI-BLAST for seven systems reveals that LESTAT exhibits increased sensitivity and specificity over PSI-BLAST in six of these systems, based on the number of true homologs detected and the number of families these homologs covered. Notably, many of the hits identified are unique to each method, presumably resulting from the distinct differences in the two approaches. Taken together, these findings suggest that LESTAT is a useful complementary method to PSI-BLAST in the detection of distant homologs.
    Proteins Structure Function and Bioinformatics 06/2008; 71(3):1409-19. · 3.34 Impact Factor

Publication Stats

77 Citations
53.38 Total Impact Points

Institutions

  • 2013–2014
    • The Chinese University of Hong Kong
      • School of Life Sciences
      Hong Kong, Hong Kong
    • Academia Sinica
      • Institute of Chemistry
      Taipei, Taipei, Taiwan
  • 2008–2013
    • The Ohio State University
      • Department of Chemistry and Biochemistry
      Columbus, Ohio, United States