Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology > Adam Barb

Adam Barb

Assistant Professor

Glycoprotein structure and immune activation, nuclear magnetic resonance spectroscopy

4210 Molecular Biology Building
Dept. of Biochemistry, Biophysics & Molecular Biology
Iowa State University
Ames, IA  50011

Phone:  (515) 294-8928


B.S. Purdue University
M.S. North Carolina State University
Ph.D. Duke University

NCBI – Complete Bibliography of Published Work – Adam Barb

Research Summary

Our laboratory pursues two independent research directions: Human antibody/receptor interactions (funded by the NIH), and a biochemistry approach towards generating biorenewable chemicals (funded through the Center for BioRenewable Chemicals (CBiRC) at ISU (an NSF ERC). We also benefited from the support of Iowa State University, the Lawrence H. Baker Center (ISU), the Center for Metabolic Biology (ISU), and the Roy J. Carver Charitable Trust.

Adam Barb - Photo 1

I.  Human antibody/receptor interactions

We made contributions in the area of immunology by describing critical structural features of immunoglobulins (antibodies) and identifying future areas for the improvement of disease treatments. Therapeutic monoclonal antibodies (mAbs) are the fastest growing class of new FDA-approved drugs that treat numerous cancers, autoimmune diseases, and transplant rejection; however, potentially grievous side effects that limit safety could be avoided by rational mAb design. Advances from our laboratory indicate mAb affinity for cell surface receptors can be increased; it is well known that increases in mAb affinity correlate with increased patient outcome. mAbs are expected to dominate the future of drug development due to their capabilities; however, mAbs are complex macromolecules and many details regarding how these drugs function remain undefined. Our primary objective is to answer basic questions about how mAbs, and thus a component of the human immune system, function to identify areas of the drug to target for improved efficacy and tolerance. The potential impact of this research is wide reaching.

We strive to determine how a conserved and necessary post-translational modification of immunoglobulins modulates the immune response. The composition of a carbohydrate chain (an N-glycan) attached to the asparagine297 residue of the IgG Fragment crystallizable (Fc) influences the strength of the body’s immune response. Shorter forms of this N-glycan are prevalent in patients with autoimmune disorders, notably rheumatoid arthritis, though it is unclear why. These same glycan forms also perturb the Fc – Fc g receptor (FcgR) interaction, though when our work in this area began, a biophysical mechanism linking the Fc glycan and immune activation had not yet been described. Forty years ago, a structure of IgG1 Fc was solved by x-ray crystallography (Huber et al. Nature 1976). Over the intervening years many similar structures have been determined, but all of these fail to offer a testable hypothesis that addresses why the Fc N-glycan composition is important. In particular, a structure of Fc in complex with the Fc g receptor IIIa (FcgRIIIa), a major inflammatory receptor, showed no direct contact between the Fc N-glycan and the FcRIIIa polypeptide (Sondermann et al. Nature 2000).

A breakthrough came in 2011 when my mentor and I published a stunning observation: though the Fc N-glycans appeared immobilized by contacts with the Fc polypeptide in images determined using x-ray crystallography, under physiologically-relevant conditions the N-glycan termini exhibit significant motion (Barb and Prestegard, Nature Chemical Biology).

As a new laboratory we tested whether Fc:FcgRIIIa affinity is modulated through motion of the Fc N-glycan, and how the composition of the Fc N-glycan directly affects motion. We determined that Fc with specific amino acid changes (mutations) at the interface between the N-glycan and polypeptide residues, distinct from the Fc/FcgRIIIa interface, showed greater N-glycan mobility (Subedi, Hanson and Barb, Structure 2014). This mobility directly correlated with FcgRIIIa affinity. Thus, as the Fc N-glycan experienced greater motion, the Fc affinity for FcgRIIIa decreased with a linear relationship. This provided direct evidence that the three-dimensional conformation of the N-glycan contributes to Fc function through an allosteric mechanism. Besides proving a novel role for N-glycans in biology, we published the first description linking Fc N-glycan conformation to function.

Our result linking N-glycan motion with receptor binding was fundamental, however, a question remained unanswered of how the conserved N-glycan affected the three dimensional structure of IgG Fc and contributed to receptor binding. One predominant hypothesis permeated the literature: The Fc N-glycan physically separates two Fc domains creating an optimal binding site for the FcgR. We found evidence to the contrary that suggested an alternative mechanism: the N-glycan affects localized Fc protein structure around the point of N-glycan attachment (asparagine297) and does not alter domain orientation. This work was published in a (Subedi and Barb, Structure 2015). As a result, we were able to directly relate the structure of the Fc to the composition of the N-glycan, and provide a powerful structure-activity link that for the first time that unambiguously links immunoglobulin structure with function.

We are continuing our work by focusing on the role of receptor N-glycosylation and an atomic-level description of the role of N-glycosylation in stabilizing the Fc polypeptide structure. Our work is pioneering and lends a high-resolution description of how glycans influence antibody structure/function and uncovers a new, potentially widespread, mechanism to control immune activation. Non-nutritive carbohydrates are found on every living cell, and the technical capability to describe structure/activity relationships is nascent.

Adam Barb - Photo 2

II.  A biochemistry approach towards generating biorenewable chemicals

The goal of this project is to generate engineered biocatalysts and develop synthetic metabolic pathways that are based on bacterial fatty acid biosynthesis but operate in parallel. This approach will permit the production of novel biorenewable chemicals in industrial fermentation applications. The parallel pathways will be used to produce novel compounds from endogenous pools of acyl-CoAs (a carbon-donating metabolite). The unique aspect of this project is that like many polyketide metabolic pathways, compounds will be built upon an acyl-carrier-protein (ACP) scaffold and enzymatically hydrolyzed to generate a free carboxylic acid. We are currently exploring the engineering potential of three E. coli fatty acid biosynthesis enzymes (holo-ACP synthase, FabD, and FabH). In so doing, we will develop a defined set of rules for modifying these enzymes that will be predictive and permit custom chemical production based upon this theoretical platform.

The primary achievements towards our goal of developing an engineered and parallel synthetic pathway include advances in protein preparation, structure determination and assay development. We established methods to prepare pure holo-, acetyl- and malonyl-ACP that are published in the journal Protein Expression and Purification (Marcella, Jing and Barb, 2015). We were able to efficiently prepare high yields of pure forms of these proteins and use them as reagents for assays with the FabD and FabH enzymes mentioned above. This material also allowed us to explore designs of the FabD enyme that exhibited dramatically broader substrate recognition than the wild-type enzyme and represents a crucial step to designing our novel parallel pathways.

An additional barrier to designing a parallel metabolic pathway has been a lack of high-resolution information about our enyzymes targets. We are addressing this by x-ray crystallography studies of the holo-ACP synthase (in our lab) and FabH enzymes (primarily in the Honzatko lab). We have had remarkable results with the holo-ACP synthase and have a high-resolution model of the enzyme at 1.8Å resolution and a diffraction map, collected in the end of February to 3.0Å, that contains reflections from the holo-ACP synthase in complex with its ACP substrate.

Our efforts to perform a precise functional characterization were limited by assays reported in the literature. Many of these relied upon radioactive nuclei and were not high-throughput. We developed a fluorescence-based assay to detect CoA formation in a FabD reaction with malonyl-CoA and holo-ACP as substrates. Currently, we are measuring the KM/kcat values for the wild type and designed enzymes with a range of synthetic CoAs produced in the lab. These values will represent a quantitative analysis of the effect of the FabD mutations on substrate specificity. The high throughput assay as proven to be robust, sensitive, flexible and adaptable to other enzymes. We characterized the activity of the E. coli FabH enzyme with the new assay and developed a platform to screen engineered FabH variants.

Select Publications

1.  The Structural Role of Antibody N-Glycosylation in Receptor Interactions.  Subedi GP, Barb AWStructure.
2015 Sep 1;23(9):1573-83. doi: 10.1016/j.str.2015.06.015. Epub 2015 Jul 23. 
PMID: 26211613 – Similar articles –
Select item 26008118

2.  Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development.  Marcella AM, Jing F,
Barb AW
Protein Expr Purif. 2015 May 22. pii: S1046-5928(15)00128-X. doi: 10.1016/j.pep.2015.05.013.  [Epub ahead of print] 
PMID: 26008118 –
Similar articles – Select item 25926001

3.  A perspective on the structure and receptor binding properties of immunoglobulin G Fc. Hanson QM, Barb AWBiochemistry.
2015 May 19;54(19):2931-42. doi: 10.1021/acs.biochem.5b00299. Epub 2015 May 7. Review. 
PMID: 25926001 –
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Select item 25551295

4.  Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-N-acetylglucosamine labeling of immunoglobulin G1.  Barb AWBiochemistry. 2015 Jan 20;54(2):313-22. doi: 10.1021/bi501380t.
Epub 2014 Dec 31. 
PMID: 25551295 Free PMC Article – Similar articles – Select item 25199692

5.  Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding.  Subedi GP,
Hanson QM, Barb AW
Structure. 2014 Oct 7;22(10):1478-88. doi: 10.1016/j.str.2014.08.002. Epub 2014 Sep 4.  PMID: 25199692 –
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Select item 24522230

6.  Immunoglobulin G1 Fc domain motions: implications for Fc engineering.  Frank M, Walker RC, Lanzilotta WN, Prestegard JH,
Barb AW
J Mol Biol. 2014 Apr 17;426(8):1799-811. doi: 10.1016/j.jmb.2014.01.011. Epub 2014 Feb 9.  PMID: 24522230 Free PMC
Article –
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7.  Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate.  Barb AW, Hekmatyar SK, Glushka JN, Prestegard
J Magn Reson. 2013 Mar;228:59-65. doi: 10.1016/j.jmr.2012.12.013. Epub 2013 Jan 4.  PMID: 23357427 Free PMC Article –
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8.  Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding.  Barb AW, Wang X, Prestegard JH.  Protein Expr Purif. 2013 Apr;88(2):183-9. doi: 10.1016/j.pep.2013.01.005. Epub 2013 Jan 12.  PMID: 23321067 Free PMC Article – Similar articles – Select item 22851279

9.  Lanthanide binding and IgG affinity construct: potential applications in solution NMR, MRI, and luminescence microscopy. 
Barb AW, Ho TG, Flanagan-Steet H, Prestegard JH. 
Protein Sci. 2012 Oct;21(10):1456-66. doi: 10.1002/pro.2133. Epub 2012 Aug 22. 
PMID: 22851279 Free PMC Article –
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10.  NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.  Barb AW, Meng L, Gao Z, Johnson RW, Moremen KW, Prestegard JH.  Biochemistry. 2012 Jun 5;51(22):4618-26. Epub 2012 May 22.  PMID: 22574931 Free PMC Article – Similar articles – Select item 22058570

11.  Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR.  Barb AW, Glushka JN, Prestegard JH.  J Chem Educ.
2011 Jan 1;88(1):95-97. 
PMID: 22058570 Free PMC Article – Similar articles – Select item 21947924

12.  NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides.  Barb AW, Freedberg DI,
Battistel MD, Prestegard JH. 
J Biomol NMR. 2011 Sep;51(1-2):163-71. doi: 10.1007/s10858-011-9550-0. Epub 2011 Sep 27.  PMID:
21947924 Free PMC Article –
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13.  Exchange facilitated indirect detection of hyperpolarized 15ND2-amido-glutamine.  Barb AW, Hekmatyar SK, Glushka JN,
Prestegard JH. 
J Magn Reson. 2011 Oct;212(2):304-10. doi: 10.1016/j.jmr.2011.07.008. Epub 2011 Jul 21.  PMID: 21824795 Free
PMC Article –
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14.  NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.  Barb AW, Prestegard JH.  Nat Chem Biol. 2011 Mar;7(3):147-53. doi: 10.1038/nchembio.511. Epub 2011 Jan 23.  PMID: 21258329 Free PMC Article – Similar articles