Leonard D. Spicer

Identification of autoantigens recognized by the 2F5 and 4E10 broadly neutralizing HIV-1 antibodies

Immunological tolerance to self-antigen impairs humoral responses to HIV-1.

Rapid debenzylation of N-benzylamino derivatives to amino-derivatives using ammonium formate as catalytic hydrogen transfer agent

Abstract Various N-benzyl derivatives of amino acids and amines were deprotected to the corresponding free amino acids and amines using ammonium formate as the hydrogen source.

High-level 2H/13C/15N labeling of proteins for NMR studies

SummaryThe protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties.

Debenzylation of N-Benzylamino Derivatives by Catalytic Transfer Hydrtyation With Ammonium Formate

Abstract A method for deprotection of several N-benzyl derivatives of amines to the corresponding amino derivatives with ammonium formate and 10% Pd-C is reported.

A refocused and optimized HNCA: increased sensitivity and resolution in large macromolecules.

SummaryA 3D optimized, refocused HNCA experiment is described. It is demonstrated to yield a dramatic increase in sensitivity when applied to [13C,15N]-labeled human carbonic anhydrase II, a 29-kDa protein. The reasons for the gain in sensitivity are discussed, and 3 distinct areas for further development are indicated.

MetJ repressor interactions with DNA probed by in-cell NMR

Atomic level characterization of proteins and other macromolecules in the living cell is challenging. Recent advances in NMR instrumentation and methods, however, have enabled in-cell studies with prospects for multidimensional spectral characterization of individual macromolecular components. We present NMR data on the in-cell behavior of the MetJ repressor from Escherichia coli, a protein that regulates the expression of genes involved in methionine biosynthesis. NMR studies of whole cells along with corresponding studies in cell lysates and in vitro preparations of the pure protein give clear evidence for extensive nonspecific interactions with genomic DNA. These interactions can provide an efficient mechanism for searching out target sequences by reducing the dependence on 3-dimensional diffusion through the crowded cellular environment. DNA provides the track for MetJ to negotiate the obstacles inherent in cells and facilitates locating and binding specific repression sites, allowing for timely control of methionine biosynthesis.

Reduction of aldehydes and ketones to methylen derivatives using ammonium formate as a catalytic hydrogen transfer agent

Abstract Various aromatic aldehydes and ketones were reduced to the corresponding hydrocarbons using ammonium formate as the hydrogen source.

Structure of an HIV-1–neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer

Significance A major roadblock in the development of an HIV vaccine is the need to develop vaccine regimens that will induce antibodies that bind to conserved regions of the HIV envelope and neutralize many different virus quasispecies. One such envelope target is at the region closest to the membrane, the glycoprotein (gp) 41 membrane proximal external region (MPER). Previous work has demonstrated that antibodies that target this region bind both to the gp41 polypeptide and to the adjacent viral membrane. However, what has been missing is a view of what the MPER-neutralizing epitopes may look like in the context of a trimeric orientation with lipids. We have constructed an MPER trimer associated with lipids and solved the trimer structure by NMR spectroscopy. The membrane proximal external region (MPER) of HIV-1 glycoprotein (gp) 41 is involved in viral–host cell membrane fusion. It contains short amino acid sequences that are binding sites for the HIV-1 broadly neutralizing antibodies 2F5, 4E10, and 10E8, making these binding sites important targets for HIV-1 vaccine development. We report a high-resolution structure of a designed MPER trimer assembled on a detergent micelle. The NMR solution structure of this trimeric domain, designated gp41-M-MAT, shows that the three MPER peptides each adopt symmetric α-helical conformations exposing the amino acid side chains of the antibody binding sites. The helices are closely associated at their N termini, bend between the 2F5 and 4E10 epitopes, and gradually separate toward the C termini, where they associate with the membrane. The mAbs 2F5 and 4E10 bind gp41-M-MAT with nanomolar affinities, consistent with the substantial exposure of their respective epitopes in the trimer structure. The traditional structure determination of gp41-M-MAT using the Xplor-NIH protocol was validated by independently determining the structure using the DISCO sparse-data protocol, which exploits geometric arrangement algorithms that guarantee to compute all structures and assignments that satisfy the data.

Crystal structure of a non-neutralizing antibody to the HIV-1 gp41 membrane-proximal external region

The monoclonal antibody 13H11 shares part of its epitope in the HIV-1 gp41 membrane-proximal external region (MPER) with the rare, broadly neutralizing human antibody 2F5. Although 13H11 partially cross-blocked 2F5 binding, 13H11 is non-neutralizing and does not block 2F5 neutralization. We show that unlike 2F5, 13H11 binds to a well-defined helical MPER structure that is consistent with the structure of gp41 in a post-fusion six-helix bundle conformation.

Magnetic field effects on surgical ligation clips

Magnetic forces exerted on surgical clips and the magnetic resonance imaging distortion they create in phantoms and rabbits at magnetic field strengths of 1.5 Tesla were investigated. Results are reported for both ligation and aneurysm clips manufactured from three types of stainless steel as well as titanium, tantalum and niobium metals. Paramagnetism and eddy currents were measured in a customized moving Gouy balance. Direct measurements of other magnetic forces were carried out in a 1.5T MRI system. The titanium and tantalum clips showed the least interaction with the magnetic field, both in terms of forces exerted and the observed image distortion with the larger clips generating the larger interactions. The strongest field distortions and attractive forces occurred with 17-7PH stainless steel clips. These interactions were ferromagnetic in origin and of sufficient strength to present significant risk to patients having this type of clip present during an MRI scan.