• Center for Macromolecular Interactions
  • CMI Laboratory

    Welcome to the CMI

    An HMS core for the characterization of macromolecules and their interactions.

  • CMI SEC-MALS system

    CMI SEC-MALS system

    Multi-Angle Light Scattering and Dynamic Light Scattering are used to measure the molar mass and hydrodynamic radius.

  • CMI SPR and BLI instruments

    CMI SPR and BLI Instruments

    Surface Plasmon Resonance and Biolayer Interferometry are used to measure binding kinetics.

  • CMI MST and ITC instruments

    CMI MST and ITC Instruments

    Isothermal Titration Calorimetry and Microscale Thermophoresis measuring binding in solution.

Coronavirus Update

The CMI is in Phase 2B Re-Entry.  The CMI COVID-19 Plan summarizes the changes implemented at CMI to maintain social distancing, enhance safety and accomodate as many users as possible. 

Center for Macromolecular Interactions

Welcome to the Center for Macromolecular Interactions (CMI) in the department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School.  Our mission is to enhance basic research in the HMS community by providing scientific consultation, training and access to shared biophysical instruments for the characterization and analysis of macromolecules and their complexes.

The facility currently includes instruments for Isothermal Titration Calorimetry (ITC)Surface Plasmon Resonance (SPR)Biolayer Interferometry (BLI)Differential Scanning Fluorimetry (DSF)Circular Dichroism (CD)Light Scattering: size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and Dynamic Light Scattering (DLS), and MicroScale Thermophoresis (MST)

Recent User Publications

Jiang H, Cole PA. N-Terminal Protein Labeling with N-Hydroxysuccinimide Esters and Microscale Thermophoresis Measurements of Protein-Protein Interactions Using Labeled Protein. Curr Protoc 2021;1(1):e14.Abstract
Protein labeling strategies have been explored for decades to study protein structure, function, and regulation. Fluorescent labeling of a protein enables the study of protein-protein interactions through biophysical methods such as microscale thermophoresis (MST). MST measures the directed motion of a fluorescently labeled protein in response to microscopic temperature gradients, and the protein's thermal mobility can be used to determine binding affinity. However, the stoichiometry and site specificity of fluorescent labeling are hard to control, and heterogeneous labeling can generate inaccuracies in binding measurements. Here, we describe an easy-to-apply protocol for high-stoichiometric, site-specific labeling of a protein at its N-terminus with N-hydroxysuccinimide (NHS) esters as a means to measure protein-protein interaction affinity by MST. This protocol includes guidelines for NHS ester labeling, fluorescent-labeled protein purification, and MST measurement using a labeled protein. As an example of the entire workflow, we additionally provide a protocol for labeling a ubiquitin E3 enzyme and testing ubiquitin E2-E3 enzyme binding affinity. These methods are highly adaptable and can be extended for protein interaction studies in various biological and biochemical circumstances. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Labeling a protein of interest at its N-terminus with NHS esters through stepwise reaction Alternate Protocol: Labeling a protein of interest at its N-terminus with NHS esters through a one-pot reaction Basic Protocol 2: Purifying the N-terminal fluorescent-labeled protein and determining its concentration and labeling efficiency Basic Protocol 3: Using MST to determine the binding affinity of an N-terminal fluorescent-labeled protein to a binding partner. Basic Protocol 4: NHS ester labeling of ubiquitin E3 ligase WWP2 and measurement of the binding affinity between WWP2 and an E2 conjugating enzyme by the MST binding assay.
Patnaik D, Pao P-C, Zhao W-N, Silva CM, Hylton NK, Chindavong PS, Pan L, Tsai L-H, Haggarty SJ. Exifone Is a Potent HDAC1 Activator with Neuroprotective Activity in Human Neuronal Models of Neurodegeneration. ACS Chem Neurosci 2021;12(2):271-284.Abstract
Genomic instability caused by a deficiency in the DNA damage response and repair has been linked to age-related cognitive decline and neurodegenerative diseases. Preventing genomic instability that ultimately leads to neuronal death may provide a broadly effective strategy to protect against multiple potential genotoxic stressors. Recently, the zinc-dependent class I histone deacetylase (HDAC1) has been identified as a critical factor for protecting neurons from deleterious effects of DNA damage in Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). Translating these observations to a novel neuroprotective therapy for AD, ALS, and FTD may be advanced by the identification of small molecules capable of increasing the deacetylase activity of HDAC1 selectively over other structurally similar HDACs. Here, we demonstrate that exifone, a drug previously shown to be effective in treating cognitive deficits associated with AD and Parkinson's disease, the molecular mechanism of which has remained poorly understood, potently activates the deacetylase activity of HDAC1. We show that exifone acts as a mixed, nonessential activator of HDAC1 that is capable of binding to both free and substrate-bound enzyme, resulting in an increased relative maximal rate of HDAC1-catalyzed deacetylation. Exifone can directly bind to HDAC1 based upon biolayer interferometry assays with kinetic and selectivity profiling, suggesting that HDAC1 is preferentially targeted compared to other class I HDACs and the kinase CDK5, which have also been implicated in neurodegeneration. Consistent with a mechanism of deacetylase activation intracellularly, the treatment of human induced pluripotent stem cell (iPSC)-derived neuronal cells resulted in globally decreased histone acetylation. Moreover, exifone treatment was neuroprotective in a tauopathy patient iPSC-derived neuronal model subject to oxidative stress. Taken together, these findings reveal exifone as a potent activator of HDAC1-mediated deacetylation, thereby offering a lead for novel therapeutic development aiming to protect genomic integrity in the context of neurodegeneration and aging.
Saundh SL, Patnaik D, Gagné S, Bishop JA, Lipsit S, Amat S, Pujari N, Nambisan AK, Bigsby R, Murphy M, Tsai L-H, Haggarty SJ, Leung AK-W. Identification and Mechanistic Characterization of a Peptide Inhibitor of Glycogen Synthase Kinase (GSK3β) Derived from the Disrupted in Schizophrenia 1 (DISC1) Protein. ACS Chem Neurosci 2020;11(24):4128-4138.Abstract
Glycogen synthase kinase 3-beta (GSK3β) is a critical regulator of several cellular pathways involved in neurodevelopment and neuroplasticity and as such is a potential focus for the discovery of new neurotherapeutics toward the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3β have been focused on developing small molecule inhibitors that compete with adenosine triphosphate (ATP) through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The disrupted in schizophrenia 1 (DISC1) protein has previously been shown to bind and inhibit GSK3β activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3β in a noncompetitive mode distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3β: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3β binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3β.
Lyons SM, Kharel P, Akiyama Y, Ojha S, Dave D, Tsvetkov V, Merrick W, Ivanov P, Anderson P. eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function. Nucleic Acids Res 2020;48(11):6223-6233.Abstract
As cells encounter adverse environmental conditions, such as hypoxia, oxidative stress or nutrient deprivation, they trigger stress response pathways to protect themselves until transient stresses have passed. Inhibition of translation is a key component of such cellular stress responses and mounting evidence has revealed the importance of a class of tRNA-derived small RNAs called tiRNAs in this process. The most potent of these small RNAs are those with the capability of assembling into tetrameric G-quadruplex (G4) structures. However, the mechanism by which these small RNAs inhibit translation has yet to be elucidated. Here we show that eIF4G, the major scaffolding protein in the translation initiation complex, directly binds G4s and this activity is required for tiRNA-mediated translation repression. Targeting of eIF4G results in an impairment of 40S ribosome scanning on mRNAs leading to the formation of eIF2α-independent stress granules. Our data reveals the mechanism by which tiRNAs inhibit translation and demonstrates novel activity for eIF4G in the regulation of translation.
Mineo M, Lyons SM, Zdioruk M, von Spreckelsen N, Ferrer-Luna R, Ito H, Alayo QA, Kharel P, Giantini Larsen A, Fan WY, Auduong S, Grauwet K, Passaro C, Khalsa JK, Shah K, Reardon DA, Ligon KL, Beroukhim R, Nakashima H, Ivanov P, Anderson PJ, Lawler SE, Chiocca AE. Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus. Mol Cell 2020;78(6):1207-1223.e8.Abstract
Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.

CMI News

New PPMS System

November 1, 2020

The CMI has migrated to a new shared instance of our PPMS booking system.  This is a major overhaul of our system, so we may encounter some issues during the transition phase. Don’t hesitate to reach out if you have any problems. 

All users will need an active CMI Instrument Access Project.

  • In the new system, you will need to select this project when you make instrument reservations or order consumables.
  • If you currently have instrument rights, then I have already created a project for you in the new system.
  • New users will need to request a project in the new PPMS system before requesting training or gaining instrument access.

CMI Getting Started Guides updated

May 22, 2020
Instrument getting started guides have all been updated with new content and a new look. Get the latest version in instrument pages in Technologies.  Please let us know if you find any problems.