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The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University

Introduction.
The Sakmar Laboratory research program focuses on the molecular mechanism of transmembrane signal transduction by a ubiquitous family of cell-surface receptors called G protein-coupled receptors (GPCRs). GPCRs are serpentine proteins that thread back and forth through the cell membrane seven times. Genes encoding GPCRs make up the largest family in the human genome. GPCRs are responsible for a myriad of signaling processes. For example, specific GPCRs in the retinal detect light and initiate visual phototransduction – color vision depends on the ability of GPCRs sense different colors of light. GPCRs are also responsible for the sense of smell (olfaction) in mammals, and for chemotaxis in lower animals such as flies and worms. A large number of hormones such as adrenaline and glucagon also bind to specific GPCRs to regulate organ physiology in the body. In fact, more than one-quarter of therapeutic drugs targeted to proteins in the human body modulated the activity of GPCRs.

Past Accomplishments.
During his post-doctoral research training at M.I.T. in the mid-1980s, Dr. Sakmar was among the first scientist to study the function of GPCRs using new techniques of molecular biology, in particular heterologous expression and site-directed mutagenesis – a combination of methods where it is possible to introduce amino acid changes at specific sites in the receptor protein and then measure the effect on function. Dr. Sakmar was the first to study mutants of rhodopsin, the receptor for dim-light in the rod cell of the retinal. He was also the first to study the effects of mutations on the ability of GPCRs to couple to another important class of cellular signaling proteins – G proteins. Dr. Sakmar has made major contributions to understanding the chemical basis for color vision. To detect light, visual GPCRs, sometimes called visual pigments, bind a unique chemical form of vitamin A. Humans have three different visual pigments, which together allow color vision. Dr. Sakmar and his colleagues, in particular the laboratory of Prof. Richard A. Mathies at University of California at Berkeley, identified the specific amino acids in each pigment that “tune” its spectral properties. Using resonance Raman spectroscopy, they also proposed a model for the physical chemistry of spectral tuning. Dr. Sakmar has also worked on the dynamics of receptor activation – the conformational changes that rapidly occur in the receptor when it absorbs a photon of light or binds to a hormone. It is these conformational changes that carry a signal across the membrane, since the photon or the hormones do not enter the interior of the cell. Dr. Sakmar and colleagues, including Prof. Henry R. Bourne at University of California at San Francisco, proposed the “helix movement model of receptor activation” and put forth the “protonation hypothesis” to explain the regulation of receptor activity. The “helix movement” model is now widely accepted as a key concept in the field of transmembrane signaling.
Computer-generated structural models of GPCRs based on the crystal structure of rhodopsin and other GPCRs have allowed Dr. Sakmar and his colleagues to study specific receptor-drug interactions with the aim of improving the design and synthesis of new drugs with higher potency and fewer side effects. For example, the virus that causes AIDS, HIV, hijacks a GPCR found on lymphocytes, the chemokine receptor 5 (CCR5), to gain entry into the cell. A new class of drugs designed to bind to CCR5 and block HIV cellular entry was recently developed using knowledge of CCR5 structure and biology. Dr. Sakmar and his colleagues, including Prof. John P. Moore at Weill-Cornell Medical College, made key contributions to identifying and optimizing drugs that could binding tightly and specifically to CCR5. The Sakmar Laboratory continues to study the structures of CCR5 and related chemokine receptors and how they become modified by cellular enzymes during biosynthesis. These studies are directly relevant to the molecular pathophysiology of AIDS, and are also relevant to stem cell biology and cancer metastasis, in which chemokine receptors also play key roles.

Interdisciplinary Science.
Studies of membrane protein dynamics require expertise is several areas of experimental biology and chemistry. Dr. Sakmar’s laboratory at Rockefeller University currently includes experts in chemical biology, biomolecular spectroscopy, computational chemistry, and molecular biology. Dr. Sakmar and his colleagues have pioneered the development of interdisciplinary experimental approaches to study the molecular mechanism of signal transduction by GPCRs. As noted earlier, with Prof. R. A. Mathies, Dr. Sakmar was the first to study expressed visual pigments by resonance Raman spectroscopy – a laser method that probes the unique vibrations between atoms in the vitamin A chromophore. He also worked with Prof. Friedrich Siebert at Ludwigs-Universität in Freiburg, Germany to provide the first infrared spectra of expressed visual pigments in which the light-dependent movement of specific amino acid groups could be detected. He also worked with Prof. David S. Kliger of University of California at Santa Cruz to study what happens in the first few nanoseconds after light activates a visual pigment. Dr. Sakmar has also worked over many years with Prof. Steven O. Smith at Stony Brook University to develop and exploit solid-state NMR approaches to obtain dynamic structural information about GPCRs in order to highlight the precise movements that occur in the protein’s interior during activation.

Recent Accomplishments.
The interdisciplinary approach of the Sakmar laboratory often results in innovative breakthroughs and new methods and strategies to address important problems in diverse fields.

Recent innovations include:
1) The adaptation of an amber codon suppression strategy to introduce unnatural amino acids (UAAs) into engineered GPCRs expressed in mammalian cells in culture, which provides a general tool to label GPCRs with fluorescent probes.

2) The invention of a novel membrane nanostructure, called NABBs (nano-scale apolipoprotein-bound bilayers), to stabilize membrane proteins in a native-like membrane environment.

3) The application of advanced computer methods, including all-atom molecular dynamics simulations and coarse-grain simulations, to the question of GPCR assembly and dynamics in membranes.

4) The discovery that a cellular protein, NucB1, which normally regulates G protein signaling and transport, can inhibit the formation of pathogenic amyloid fibrils by capping smaller growing protofibrils.

Looking Ahead.
For nearly 20 years, the Sakmar Laboratory has been a productive center for innovative studies of GPCRs and related fields, and has also served as a training ground for students at all levels of their scientific training, from post-doctoral fellows to high school students. Working together in the future, members of the Sakmar Laboratory hope to reveal with chemical precision how organisms sense their environment and how cells use chemical signals to communicate. Their studies are directly relevant to drug discovery and to improving therapy for a number of human diseases – AIDS, visual disorders such as retinitis pigmentosa and age-related macular degeneration, and Alzheimer’s disease and other amyloid diseases.


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University

This Month's Highlight:

G Protein Crystal Structure Sheds Light on Mechanism of GDP Release

Heptahelical G protein-coupled receptors (GPCRs) comprise the largest family of cell-surface receptors and are the largest target class for pharmaceutical agents. Recently, crystal structures of several GPCRs were published in Nature, Science and other leading journals. The next frontier is to elucidate how receptors coupled to heterotrimeric G proteins to switch them from “off” to “on” in response to a signal. What movements and conformational changes cause G proteins to release GDP and take up GTP?
To address these questions, Neeraj Kapoor, a graduate student in the Tri-Institutional Program in Chemical Biology solved the crystal structure of an interesting mutant G protein that exhibits a very high intrinsic rate of basal nucleotide exchange. The report of his work, published on-line in Journal of Molecular Biology, presents a detailed biochemical and biophysical analysis of an expressed Gi mutant that bears directly on the question of how the nucleotide-binding pocket of the G protein is coupled to the ligand-binding pocket of a GPCR at a distance of ~50-70 Å. Neeraj’s work, which was carried out with Sakmar Laboratory members Santosh Menon, Pallavi Sachdev and collaborator Radha Chauhan, extents far beyond earlier mutagenesis and structural studies of G proteins and suggests a plausible mechanistic model describing the precise steps in a sequence of conformational changes that cause GDP release. Interestingly, the model shows striking similarities between nucleotide release by heterotrimeric G proteins and small G proteins – a finding that may have been anticipated, but was not documented earlier. This work, which ties together mechanistic studies of GPCR- and small G-protein signaling, will be of interest to a broad group of researchers.


Structural Evidence for a Sequential Release Mechanism for Activation of Heterotrimeric G Proteins.
Kapoor N, Menon ST, Chauhan R, Sachdev P, Sakmar TP.
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Heptahelical G protein-coupled receptors (GPCRs) couple to heterotrimeric G proteins to relay extracellular signals to intracellular signaling networks, but the molecular mechanism underlying GDP release by the G protein alpha-subunit is not well understood. Amino acid substitutions in the conserved alpha5 helix of G(i), which extends from the C-terminal region to the nucleotide-binding pocket, cause dramatic increases in basal (receptor-independent) GDP release rates. For example, mutant Galpha(i1)-T329A shows an 18-fold increase in basal GDP release rate, and when expressed in culture it causes a significant decrease in forskolin-stimulated cAMP accumulation. The crystal structure of Galpha(i1)-T329A*GDP shows substantial conformational rearrangement of the switch I region and additional striking alterations of side chains lining the catalytic pocket that disrupt the Mg(+2) coordination sphere and dislodge bound Mg(+2). We propose a "sequential release" mechanism whereby a transient conformational change in the alpha5 helix alters switch I to induce GDP release. Interestingly, this mechanistic model for heterotrimeric G protein activation is similar to that suggested for the activation of the plant small G protein Rop4 by RopGEF8.

[PMID: 19703466 [PubMed - as supplied by publisher]


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Rockefeller University

Laboratory of Molecular Biology & Biochemistry
1230 York Avenue
New York, NY 10065

212-327-8284 tel

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picThomas P. Sakmar, M.D.
Professor
Head of Laboratory


SAKMAR AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 187
Room: RRB 515
Building: Rockefeller Research Building
Floor: 5

212-327-8288 tel.

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picMarguerite Mangin
Senior Research Associate
Academic Programs Director


MANGINM AT rockefeller DOT edu

Department: Presidents Office
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Room: CH 204
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212-327-8081 tel.

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picThomas Huber, M.D.-Ph.D.
Research Associate



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Department: Sakmar Laboratory
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212-327-8279 alt.

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picPallavi Sachdev, Ph.D.
Research Associate



SACHDEP AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 187
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

212-327-8284 tel.

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picThomas Haines, Ph.D.
Visiting Professor
Member of the Adjunct Faculty


THAINES AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 187
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

212-327-8284 tel.

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picManija Kazmi
Research Specialist



KAZMIM AT rockefeller DOT edu

Department: Sakmar Laboratory

Mail Box No: 187
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

212-327-8284 tel.

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picRuchi Gupta
Postdoctoral Associate



RGUPTA AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 187
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Building: Rockefeller Research Building
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212-327-8284 tel.

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picSaranga Naganathan
Post Doctoral Associate



SNAGANATHA AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 187
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

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picParag Mukhopadhyay
Postdoctoral Associate



PMUKHOPADHYAY AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 187
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

212-327-8284 tel.

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picNeeraj Kapoor
Graduate Fellow in Chemical Biology


NKAPOOR AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 148
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

212-327-8284 tel.

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picAmy Grunbeck
Graduate Fellow in Chemical Biology


AGRUNBECK AT rockefeller DOT edu

Department: Sakmar Laboratory
Mail Box No: 19
Room: RRB 510
Building: Rockefeller Research Building
Floor: 5

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picAdam Knepp

Graduate Fellow


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Department: Deans Office

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Room: FRS 105
Building: Founders Hall
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Web Site Issues: sysadmin


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University

PLEASE NOTE THAT ALL OF THE MATERIAL IN THIS SECTION IS COPYRIGHT PROTECTED.

To use any of our images, photographs, or movies please request permission.


Portraits Gallery
The Portraits gallery contains albums of Sakmar Lab member portraits.

The Group Shots Gallery contatins group shots and and annual lab postcards designed and produced by Karina Åberg Sakmar. Group photos were taken by Lubos Stepanek.

Workshops & Seminars Gallery
The Workshops & Seminars gallery contains albums of photographs of Sakmar Lab members presenting their work at retreats, conferences, meetings and symposia.

Laboratory Gallery
The Laboratory Gallery consists of a collection of albums containing photographs of white-board notes, schematic diagrams, laboratory equipments and other laboratory imagery. This section is password protected. Please write the Lab Manager for permission if you need access to this gallery.

Social Activities Gallery
Albums in the Social Activities Gallery include graduation pictures, party and event announcements and photos and science-related travel photos that laboratory members choose to share.


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University

PLEASE NOTE THAT ALL OF THE MATERIAL IN THIS SECTION IS COPYRIGHT PROTECTED.

To use any of our images, photographs, or movies please request permission.


Molecules Gallery
The Molecules Gallery contains a collection of albums of graphic visualizations of molecules under study or used by Sakmar Lab members.

Structural Biology Gallery
The Structural Biology Gallery contains albums of images that represent molecular structures solved and reported by Sakmar Lab members.

Receptors Gallery
The Receptors Gallery contains albums that focus on images of receptors under study in the Sakmar Lab, including rhodopsins, CCR5/CXCR4, adrenergic receptors, dopamine D4 receptor and glucagon receptor.


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University

PLEASE NOTE THAT ALL OF THE MATERIAL IN THIS SECTION IS COPYRIGHT PROTECTED.

To use any of our images, photographs, or movies please request permission.



The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry>
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry>
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University


The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University

PLEASE NOTE THAT ALL OF THE MATERIAL IN THIS SECTION IS COPYRIGHT PROTECTED.

To use any of our images, photographs, or movies please request permission.



The Sakmar Laboratory
The Laboratory of
Molecular Biology & Biochemistry
at Rockefeller University