Benjamin McFarland | Seattle Pacific University (original) (raw)

Papers by Benjamin McFarland

Advances in Protein Chemistry, 2004

NK cells are crucial components of the innate immune system, capable of directly eliminating infe... more NK cells are crucial components of the innate immune system, capable of directly eliminating infected or tumorigenic cells and regulating downstream adaptive immune responses. Unlike T cells, where the key recognition event driving activation is mediated by the unique T cell receptor (TCR) expressed on a given cell, NK cells express multiple activating and inhibitory cell-surface receptors (NKRs), often with overlapping ligand specificities. NKRs display two ectodomain structural homologies, either immunoglobulin-or C-type lectin-like (CTLD). The CTLD immunoreceptor NKG2D is found on NK cells but is also widely expressed on T cells and other immune system cells, providing stimulatory or costimulatory signals. NKG2D drives target cell killing following engagement of diverse, conditionally expressed MHC class I-like protein ligands whose expression can signal cellular distress due to infection or transformation. The symmetric, homodimeric receptor interacts with its asymmetric, monomeric ligands in similar 2:1 complexes, with an equivalent surface on each NKG2D monomer binding extensively and intimately to distinct, structurally divergent surfaces on the ligands. Thus, NKG2D ligand-binding site recognition is highly degenerate, further demonstrated by NKG2D's ability to simultaneously accommodate multiple non-conservative allelic or isoform substitutions in the ligands. In TCRs, 'induced-fit' recognition explains cross-reactivity, but structural, computational, thermodynamic and kinetic analyses of multiple NKG2D-ligand pairs show that rather than classical 'induced-fit' binding, NKG2D degeneracy is achieved using distinct interaction mechanisms at each rigid interface: recognition degeneracy by 'rigid adaptation'. While likely forming similar complexes with their ligand (HLA-E), other NKG2x NKR family members do not require such recognition degeneracy.

Immunity, Dec 1, 2003

poulos et al., 2002b), H60 (O'Callaghan et al., 2001) and possibly Mill1 and 2 (proposed on the b... more poulos et al., 2002b), H60 (O'Callaghan et al., 2001) and possibly Mill1 and 2 (proposed on the basis of homology to MIC; Kasahara et al., 2002). While murine (muNKG2D) Summary and human NKG2D (huNKG2D) are highly conserved (69% identical in the ectodomains), and the NKG2D liThe homodimeric immunoreceptor NKG2D drives the gands are well conserved within families (ranging from activation of effector cells following engagement of 55% identity among the ULBPs to greater than 84% diverse, conditionally expressed MHC class I-like probetween MIC-A and-B alleles), the ligands are distantly tein ligands. NKG2D recognition is highly degenerate related between families in terms of both sequence (23% in that a single surface on receptor monomers binds to 27% identical) and detailed structure. pairs of distinct surfaces on each structurally diver-Crystal structures are available for muNKG2D and gent ligand, simultaneously accommodating multiple huNKG2D, free (McFarland et al., 2003; Wolan et al., nonconservative ligand allelic or isoform substitu-2001) or bound to either MIC-A (allele 001; Li et al., 2001), tions. In contrast to TCR-pMHC and other NK recep-ULBP3 (Radaev et al., 2001), or RAE-1␤ (Li et al., 2002). tor-ligand interactions, thermodynamic and kinetic These structures show the homodimeric receptor interanalyses of four NKG2D-ligand pairs (MIC-A*001, MICacting with monomeric ligands in similar 2:1 complexes, B*005, ULBP1, and RAE-1␤) reported here show that with an equivalent surface on each NKG2D monomer the relative enthalpic and entropic terms, heat capacbinding intimately to pairs of distinct surfaces on the ity, association rates, and activation energy barriers asymmetric ligands, on either the ␣1 or ␣2 domains, are comparable to typical, rigid protein-protein inyielding six distinct receptor-ligand interfaces. The footteractions. Rather than "induced-fit" binding, NKG2D print and overall arrangement of the NKG2D-ligand comdegeneracy is achieved using distinct interaction plexes is strongly reminiscent of ␣␤ TCR-peptide-MHC mechanisms at each rigid interface. class I (TCR-pMHC) complexes, though the NKG2Dmediated recognition events are tighter, more extensive, Introduction more shape-complementary, and involve the C-type lectin-like domains of NKG2D rather than the immunoglob-The C-type lectin-like immunoreceptor NKG2D is an ulin-like domains of TCRs. Where MHC class I polymoroutlier member of the NKG2x-CD94 family (x ϭ A, B, C, phic and antigenic peptide sequence differences in the E, and H) in terms of sequence, distribution, multimeric binding footprint result directly in differential recognition state, and function. NKG2D was first functionally defined by the constellation of TCRs in a host, NKG2D recognias an activating receptor on natural killer (NK) cells medition conversely tolerates dramatic allelic (MIC) and isoating responses to the stress-induced expression of the form (ULBP and RAE-1) differences in contact residues MIC family of MHC class I-like ligands on intestinal epiwith only minor changes in affinity (McFarland et al., thelial cells (Bauer et al., 1999). Subsequently, NKG2D 2003; O'Callaghan et al., 2001; Steinle et al., 2001; has been shown to be broadly expressed, playing addi-Strong, 2002). tional roles in innate immunity by driving the activation This extreme recognition degeneracy (␣1 versus ␣2 of macrophages (Diefenbach et al., 2000) and particular on a ligand protein, one ligand protein versus another, ␥␦ T cell populations (Das et al., 2001), and in adaptive surfaces remodeled through substitution) is not accomimmunity by providing costimulatory signals to CD8 ϩ ␣␤ plished through dominance of electrostatic or hydropho-T cells (Groh et al., 2001). NKG2D engagement initiates bic interactions, which are relatively insensitive to precise perforin-mediated cytolytic responses against virally ingeometry and therefore enable degenerate binding, exemfected and tumorigenic cells (Cerwenka and Lanier, plified by chemokine-receptor interactions (Alexander 2001; Hayakawa et al., 2002). NKG2D is therefore a poet al., 2002; Skelton et al., 1999) and in the binding tent mediator of antiviral and antitumor immunosurveilof human growth hormone to hormone and prolactin lance. Tumor cells can evade this immunosurveillance receptors (Clackson and Wells, 1995; Somers et al., by producing soluble MIC ligands, shed through proteol-1994). Solvent is also cleanly excluded from the interysis, resulting in downregulation of NKG2D cell surface faces, disallowing variable water-mediated contacts as a means for achieving degeneracy. Conformational plasexpression on immune effector cells (Groh et al., 2002; ticity, or "induced-fit" recognition, where a flexible bind-Salih et al., 2002). To accomplish these functions, ing site can mold to accommodate diverse ligands, is NKG2D interacts with a diverse array of distinct MHC typical of many antibody-antigen interactions (James et al., 2003; Sundberg and Mariuzza, 2000) and has been

Journal of Chemical Education, Apr 18, 2011

We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatil... more We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5-12 new techniques learned, which are transferrable to graduate research in academia and industry.

Journal of the American Chemical Society, Nov 13, 1999

Immunological Reviews, Dec 1, 1999

Determination of the crystal structure of class II: peptide complexes has shown that in addition ... more Determination of the crystal structure of class II: peptide complexes has shown that in addition to pocket interactions involving the side chains of the peptide, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds which are contributed by genetically conserved amino acid residues in the class II molecule to the main chain of the peptide. Our experiments have revealed an unexpectedly large contribution of hydrogen bonds at the periphery of the MHC peptide binding pocket to MHC class II function. Kinetic studies have shown that peptide dissociation rates are profoundly accelerated by loss of a single hydrogen bonding residue. The magnitude of the effects seen with the loss in potential for a single hydrogen bond support a co-operative model in which individual bonds between class II and peptide are dependent on the integrity of neighboring interactions. Collectively our studies have revealed that MHC class II structure, peptide binding and intracellular trafficking events are critically dependent on the integrity of the hydrogen bonding network between class II molecules and its bound peptide.

Proceedings of the National Academy of Sciences of the United States of America, Jul 24, 2001

Comparison of crystallized MHC class II⅐peptide complexes has revealed that, in addition to pocke... more Comparison of crystallized MHC class II⅐peptide complexes has revealed that, in addition to pocket interactions involving the peptide side chains, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds between genetically conserved amino acid residues in the class II molecule and the main chain of the peptide. Many class II⅐peptide structures have two sets of symmetrical hydrogen bonds at the opposite ends of the class II antigen-binding groove (␤-His-81, ␤-Asn-82 vs. ␣-His-68, ␣-Asn-69). In this study, we alter these peripheral hydrogen bonds and measure the apparent contribution of each to the kinetic stability of peptide⅐class II complexes. Single conservative amino substitutions were made in the I-A d protein to eliminate participation as a hydrogen bonding residue, and the kinetic stability of a diverse set of peptides bound to the substituted I-A d proteins was measured. Although each hydrogen bond does contribute to peptide binding, our results point to the striking conclusion that those hydrogen bonds localized to the amino terminus of the peptide contribute profoundly and disproportionately to the stability of peptide interactions with I-A d. We suggest that the peripheral hydrogen bonds at the amino terminus of the bound peptide that are conserved in all class II⅐peptide crystal structures solved thus far form a cooperative network that critically regulates peptide dissociation from the class II molecule.

Journal of Molecular Biology, Jul 1, 2005

The complexity of the interaction between major histocompatibility complex class II (MHC II) prot... more The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A d-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A d MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (!5 Å).

Molecular Immunology, 2011

The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ... more The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ligands polyspecifically, yet without central conformational flexibility. Crystal structures of multiple NKG2D-ligand interactions have identified the NKG2D tyrosine pair Tyr 152 and Tyr 199 as forming multiple specific but diverse interactions with MICA and related proteins. Here we systematically altered each tyrosine to tryptophan, phenylalanine, isoleucine, leucine, valine, serine, and alanine to measure the effect of mutation on affinity and thermodynamics for binding a range of similar ligands: MICA, the higher-affinity ligand MICB, and MICdesign, a high-affinity version of MICA that shares all NKG2D contact residues with MICA. Affinity and residue size were related: tryptophan could often substitute for tyrosine without loss of affinity; loss of the tyrosine hydroxyl through mutation to phenylalanine was tolerated more at position 152 than 199; and the smallest residues coincide with lowest affinities in general. NKG2D mutant van't Hoff binding thermodynamics generally show that substitution of other residues for tyrosine causes a moderate positive or flat van't Hoff slope consistent with moderate loss of binding enthalpy. One set of NKG2D mutations caused MICA to adopt a positive van't Hoff slope corresponding to absorption of heat, and another set caused MICB to adopt a negative slope of greater heat release than wild-type. MICdesign shared one example of the first set with MICA and one of the second set with MICB. When the NKG2D mutation affinities were arranged according to change in nonpolar surface area and compared to results from specific antibody-antigen and protein-peptide interactions, it was found that hydrophobic surface loss in NKG2D reduced binding affinity less than reported in the other contexts. The hydrophobic effect at the center of the NKG2D binding appears more similar to that at the periphery of an antibodyantigen binding site than at its center. Therefore the polyspecific NKG2D binding site is more tolerant of structural alteration in general than either an antibody-antigen or protein-peptide binding site, and this tolerance may adapt NKG2D to a broad range of protein surfaces with micromolar affinity.

ChemInform, May 21, 2010

Binding Interactions Between Peptides and Proteins of the Class II Major Histocompatibility Compl... more Binding Interactions Between Peptides and Proteins of the Class II Major Histocompatibility Complex-[253 refs.].-(MCFARLAND,

Journal of Immunology, Apr 1, 2009

Immune responses are largely governed by tight, highly specific protein-protein interactions. Thi... more Immune responses are largely governed by tight, highly specific protein-protein interactions. This study focused on one such interaction between the homodimeric immunoreceptor NKG2D, found on natural killer cells, and its MHC-like ligands, which are expressed on stressed cells. Previous research has shown that the NKG2D-ligand interface structure is dominated by 4 central tyrosine residues at positions 152 and 199. We mutated these residues to smaller and larger amino acids in order to investigate the role of the tyrosines in NKG2D-ligand complexes. Eight tyrosine mutants were tested for binding to MICA, MICB and a high-affinity MICA mutant using surface plasmon resonance. For example, mutating Y199 to phenylalanine had a larger detrimental effect on binding than did Y152F, implying that the former tyrosine is more sensitive to the removal of the hydroxyl. Alanine mutations were most detrimental to complex affinity but we were still able to detect binding. We found the vant Hoff enthalpies of each mutant NKG2D-ligand interaction. We correlated observed binding energies with those predicted by a fixed-backbone RosettaDesign mutation simulation. Even simple fixed-backbone RosettaDesign provided qualitative agreement with results for 14 of 15 mutant NKG2D-ligand interactions, and quantitative agreement for 11 of those. To complete this study we plan to investigate the hydrophobic effect in the center of the site by making an additional 3 mutations (I,L, and V) at each residue.

Journal of Immunology, Apr 1, 2009

Biochemistry, Nov 24, 1999

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are ... more Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

ABSTRACT.-From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities e... more ABSTRACT.-From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities experienced unprecedented synergy. Scientific societies founded in the late eighteenth and early nineteenth century, such as the American Philosophical Society, the Academy of Natural Sciences of ...

Perspectives on Science and Christian Faith

Journal of Immunology, 2009

From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities experienced ... more From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities experienced unprecedented synergy. Scientific societies founded in the late eighteenth and early nineteenth century, such as the American Philosophical Society, the Academy of Natural Sciences of Philadelphia, and the Franklin Institute, reached a stage of broad expansion. Medical science and education ballooned, and magazines, newspapers, and publishers were proliferating. The circle of the educated elite in Philadelphia was large enough to exhibit wide professional diversity, yet small enough to allow frequent associations among its members, whether scientists, artists, or both. We propose that a daguerreotype that has appeared since 1937 in several articles, often without attribution, was taken by Paul Beck Goddard in the winter of 1842-43 in the Academy of Natural Science of Philadelphia's recently constructed building at the corner of Broad and Sansom Streets. Goddard had announced as early...

ACS Symposium Series

One educational problem that technology can address is how to facilitate authentic research exper... more One educational problem that technology can address is how to facilitate authentic research experiences in the classroom for large numbers of students. The collaborative online platform GENI (Guiding Education through Novel Investigation, found at geni-science.org) provides an application that transfers protocols among institutions and into undergraduate teaching laboratories, then collects the data from students for analysis and publication. I have used this tool for several years to conduct bioinformatics research with three or four 16-student lab sections in Biochemistry I and to prepare recombinant proteins with two or three 16-student lab sections in Biochemistry II. These projects also feed into a Survey of Physical Chemistry course. Here I present technical details of how research projects have transitioned into the teaching lab. Others in the GENI consortium have accomplished similar projects in molecular biology and genetics contexts. Education researchers on our team are developing and applying assessments to shape our use of GENI as a tool. Altogether, these show that online lab protocols and data collection can be an effective way to teach students through the creative and exploratory process of asking and answering research questions.

ACS Symposium Series

In the laboratory for Survey of Physical Chemistry, students proceeded through a five-week projec... more In the laboratory for Survey of Physical Chemistry, students proceeded through a five-week project in which they measured protein-protein binding. This project engaged the students in learning physical chemistry and laboratory teachniques as they took ownership of a particular, novel protein-protein interaction. First students purified new proteins by size-exclusion chromatography and learned about separation and diffusion. Then students measured the binding strength of new protein-protein combinations using surface plasmon resonance (SPR) as they learned about SPR physics, experimental design, equilibrium binding, and data fitting using integrated rate laws. The web-based platform GENI provided protocols to the students and collected data, organizing projects spanning multiple classes. In the space of an academic year, students asked a question, then found the answer in the lab. Together, by expressing new proteins and measuring binding thermodynamics and kinetics, we found that the NKG2D immunoreceptor and its MIC ligand proteins show remarkable cross-reactivity among human, rabbit, and gorilla orthologs.

Advances in Protein Chemistry, 2004

NK cells are crucial components of the innate immune system, capable of directly eliminating infe... more NK cells are crucial components of the innate immune system, capable of directly eliminating infected or tumorigenic cells and regulating downstream adaptive immune responses. Unlike T cells, where the key recognition event driving activation is mediated by the unique T cell receptor (TCR) expressed on a given cell, NK cells express multiple activating and inhibitory cell-surface receptors (NKRs), often with overlapping ligand specificities. NKRs display two ectodomain structural homologies, either immunoglobulin-or C-type lectin-like (CTLD). The CTLD immunoreceptor NKG2D is found on NK cells but is also widely expressed on T cells and other immune system cells, providing stimulatory or costimulatory signals. NKG2D drives target cell killing following engagement of diverse, conditionally expressed MHC class I-like protein ligands whose expression can signal cellular distress due to infection or transformation. The symmetric, homodimeric receptor interacts with its asymmetric, monomeric ligands in similar 2:1 complexes, with an equivalent surface on each NKG2D monomer binding extensively and intimately to distinct, structurally divergent surfaces on the ligands. Thus, NKG2D ligand-binding site recognition is highly degenerate, further demonstrated by NKG2D's ability to simultaneously accommodate multiple non-conservative allelic or isoform substitutions in the ligands. In TCRs, 'induced-fit' recognition explains cross-reactivity, but structural, computational, thermodynamic and kinetic analyses of multiple NKG2D-ligand pairs show that rather than classical 'induced-fit' binding, NKG2D degeneracy is achieved using distinct interaction mechanisms at each rigid interface: recognition degeneracy by 'rigid adaptation'. While likely forming similar complexes with their ligand (HLA-E), other NKG2x NKR family members do not require such recognition degeneracy.

Immunity, Dec 1, 2003

poulos et al., 2002b), H60 (O'Callaghan et al., 2001) and possibly Mill1 and 2 (proposed on the b... more poulos et al., 2002b), H60 (O'Callaghan et al., 2001) and possibly Mill1 and 2 (proposed on the basis of homology to MIC; Kasahara et al., 2002). While murine (muNKG2D) Summary and human NKG2D (huNKG2D) are highly conserved (69% identical in the ectodomains), and the NKG2D liThe homodimeric immunoreceptor NKG2D drives the gands are well conserved within families (ranging from activation of effector cells following engagement of 55% identity among the ULBPs to greater than 84% diverse, conditionally expressed MHC class I-like probetween MIC-A and-B alleles), the ligands are distantly tein ligands. NKG2D recognition is highly degenerate related between families in terms of both sequence (23% in that a single surface on receptor monomers binds to 27% identical) and detailed structure. pairs of distinct surfaces on each structurally diver-Crystal structures are available for muNKG2D and gent ligand, simultaneously accommodating multiple huNKG2D, free (McFarland et al., 2003; Wolan et al., nonconservative ligand allelic or isoform substitu-2001) or bound to either MIC-A (allele 001; Li et al., 2001), tions. In contrast to TCR-pMHC and other NK recep-ULBP3 (Radaev et al., 2001), or RAE-1␤ (Li et al., 2002). tor-ligand interactions, thermodynamic and kinetic These structures show the homodimeric receptor interanalyses of four NKG2D-ligand pairs (MIC-A*001, MICacting with monomeric ligands in similar 2:1 complexes, B*005, ULBP1, and RAE-1␤) reported here show that with an equivalent surface on each NKG2D monomer the relative enthalpic and entropic terms, heat capacbinding intimately to pairs of distinct surfaces on the ity, association rates, and activation energy barriers asymmetric ligands, on either the ␣1 or ␣2 domains, are comparable to typical, rigid protein-protein inyielding six distinct receptor-ligand interfaces. The footteractions. Rather than "induced-fit" binding, NKG2D print and overall arrangement of the NKG2D-ligand comdegeneracy is achieved using distinct interaction plexes is strongly reminiscent of ␣␤ TCR-peptide-MHC mechanisms at each rigid interface. class I (TCR-pMHC) complexes, though the NKG2Dmediated recognition events are tighter, more extensive, Introduction more shape-complementary, and involve the C-type lectin-like domains of NKG2D rather than the immunoglob-The C-type lectin-like immunoreceptor NKG2D is an ulin-like domains of TCRs. Where MHC class I polymoroutlier member of the NKG2x-CD94 family (x ϭ A, B, C, phic and antigenic peptide sequence differences in the E, and H) in terms of sequence, distribution, multimeric binding footprint result directly in differential recognition state, and function. NKG2D was first functionally defined by the constellation of TCRs in a host, NKG2D recognias an activating receptor on natural killer (NK) cells medition conversely tolerates dramatic allelic (MIC) and isoating responses to the stress-induced expression of the form (ULBP and RAE-1) differences in contact residues MIC family of MHC class I-like ligands on intestinal epiwith only minor changes in affinity (McFarland et al., thelial cells (Bauer et al., 1999). Subsequently, NKG2D 2003; O'Callaghan et al., 2001; Steinle et al., 2001; has been shown to be broadly expressed, playing addi-Strong, 2002). tional roles in innate immunity by driving the activation This extreme recognition degeneracy (␣1 versus ␣2 of macrophages (Diefenbach et al., 2000) and particular on a ligand protein, one ligand protein versus another, ␥␦ T cell populations (Das et al., 2001), and in adaptive surfaces remodeled through substitution) is not accomimmunity by providing costimulatory signals to CD8 ϩ ␣␤ plished through dominance of electrostatic or hydropho-T cells (Groh et al., 2001). NKG2D engagement initiates bic interactions, which are relatively insensitive to precise perforin-mediated cytolytic responses against virally ingeometry and therefore enable degenerate binding, exemfected and tumorigenic cells (Cerwenka and Lanier, plified by chemokine-receptor interactions (Alexander 2001; Hayakawa et al., 2002). NKG2D is therefore a poet al., 2002; Skelton et al., 1999) and in the binding tent mediator of antiviral and antitumor immunosurveilof human growth hormone to hormone and prolactin lance. Tumor cells can evade this immunosurveillance receptors (Clackson and Wells, 1995; Somers et al., by producing soluble MIC ligands, shed through proteol-1994). Solvent is also cleanly excluded from the interysis, resulting in downregulation of NKG2D cell surface faces, disallowing variable water-mediated contacts as a means for achieving degeneracy. Conformational plasexpression on immune effector cells (Groh et al., 2002; ticity, or "induced-fit" recognition, where a flexible bind-Salih et al., 2002). To accomplish these functions, ing site can mold to accommodate diverse ligands, is NKG2D interacts with a diverse array of distinct MHC typical of many antibody-antigen interactions (James et al., 2003; Sundberg and Mariuzza, 2000) and has been

Journal of Chemical Education, Apr 18, 2011

We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatil... more We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5-12 new techniques learned, which are transferrable to graduate research in academia and industry.

Journal of the American Chemical Society, Nov 13, 1999

Immunological Reviews, Dec 1, 1999

Determination of the crystal structure of class II: peptide complexes has shown that in addition ... more Determination of the crystal structure of class II: peptide complexes has shown that in addition to pocket interactions involving the side chains of the peptide, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds which are contributed by genetically conserved amino acid residues in the class II molecule to the main chain of the peptide. Our experiments have revealed an unexpectedly large contribution of hydrogen bonds at the periphery of the MHC peptide binding pocket to MHC class II function. Kinetic studies have shown that peptide dissociation rates are profoundly accelerated by loss of a single hydrogen bonding residue. The magnitude of the effects seen with the loss in potential for a single hydrogen bond support a co-operative model in which individual bonds between class II and peptide are dependent on the integrity of neighboring interactions. Collectively our studies have revealed that MHC class II structure, peptide binding and intracellular trafficking events are critically dependent on the integrity of the hydrogen bonding network between class II molecules and its bound peptide.

Proceedings of the National Academy of Sciences of the United States of America, Jul 24, 2001

Comparison of crystallized MHC class II⅐peptide complexes has revealed that, in addition to pocke... more Comparison of crystallized MHC class II⅐peptide complexes has revealed that, in addition to pocket interactions involving the peptide side chains, peptide binding to MHC class II molecules is characterized by a series of hydrogen bonds between genetically conserved amino acid residues in the class II molecule and the main chain of the peptide. Many class II⅐peptide structures have two sets of symmetrical hydrogen bonds at the opposite ends of the class II antigen-binding groove (␤-His-81, ␤-Asn-82 vs. ␣-His-68, ␣-Asn-69). In this study, we alter these peripheral hydrogen bonds and measure the apparent contribution of each to the kinetic stability of peptide⅐class II complexes. Single conservative amino substitutions were made in the I-A d protein to eliminate participation as a hydrogen bonding residue, and the kinetic stability of a diverse set of peptides bound to the substituted I-A d proteins was measured. Although each hydrogen bond does contribute to peptide binding, our results point to the striking conclusion that those hydrogen bonds localized to the amino terminus of the peptide contribute profoundly and disproportionately to the stability of peptide interactions with I-A d. We suggest that the peripheral hydrogen bonds at the amino terminus of the bound peptide that are conserved in all class II⅐peptide crystal structures solved thus far form a cooperative network that critically regulates peptide dissociation from the class II molecule.

Journal of Molecular Biology, Jul 1, 2005

The complexity of the interaction between major histocompatibility complex class II (MHC II) prot... more The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A d-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A d MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (!5 Å).

Molecular Immunology, 2011

The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ... more The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ligands polyspecifically, yet without central conformational flexibility. Crystal structures of multiple NKG2D-ligand interactions have identified the NKG2D tyrosine pair Tyr 152 and Tyr 199 as forming multiple specific but diverse interactions with MICA and related proteins. Here we systematically altered each tyrosine to tryptophan, phenylalanine, isoleucine, leucine, valine, serine, and alanine to measure the effect of mutation on affinity and thermodynamics for binding a range of similar ligands: MICA, the higher-affinity ligand MICB, and MICdesign, a high-affinity version of MICA that shares all NKG2D contact residues with MICA. Affinity and residue size were related: tryptophan could often substitute for tyrosine without loss of affinity; loss of the tyrosine hydroxyl through mutation to phenylalanine was tolerated more at position 152 than 199; and the smallest residues coincide with lowest affinities in general. NKG2D mutant van't Hoff binding thermodynamics generally show that substitution of other residues for tyrosine causes a moderate positive or flat van't Hoff slope consistent with moderate loss of binding enthalpy. One set of NKG2D mutations caused MICA to adopt a positive van't Hoff slope corresponding to absorption of heat, and another set caused MICB to adopt a negative slope of greater heat release than wild-type. MICdesign shared one example of the first set with MICA and one of the second set with MICB. When the NKG2D mutation affinities were arranged according to change in nonpolar surface area and compared to results from specific antibody-antigen and protein-peptide interactions, it was found that hydrophobic surface loss in NKG2D reduced binding affinity less than reported in the other contexts. The hydrophobic effect at the center of the NKG2D binding appears more similar to that at the periphery of an antibodyantigen binding site than at its center. Therefore the polyspecific NKG2D binding site is more tolerant of structural alteration in general than either an antibody-antigen or protein-peptide binding site, and this tolerance may adapt NKG2D to a broad range of protein surfaces with micromolar affinity.

ChemInform, May 21, 2010

Binding Interactions Between Peptides and Proteins of the Class II Major Histocompatibility Compl... more Binding Interactions Between Peptides and Proteins of the Class II Major Histocompatibility Complex-[253 refs.].-(MCFARLAND,

Journal of Immunology, Apr 1, 2009

Immune responses are largely governed by tight, highly specific protein-protein interactions. Thi... more Immune responses are largely governed by tight, highly specific protein-protein interactions. This study focused on one such interaction between the homodimeric immunoreceptor NKG2D, found on natural killer cells, and its MHC-like ligands, which are expressed on stressed cells. Previous research has shown that the NKG2D-ligand interface structure is dominated by 4 central tyrosine residues at positions 152 and 199. We mutated these residues to smaller and larger amino acids in order to investigate the role of the tyrosines in NKG2D-ligand complexes. Eight tyrosine mutants were tested for binding to MICA, MICB and a high-affinity MICA mutant using surface plasmon resonance. For example, mutating Y199 to phenylalanine had a larger detrimental effect on binding than did Y152F, implying that the former tyrosine is more sensitive to the removal of the hydroxyl. Alanine mutations were most detrimental to complex affinity but we were still able to detect binding. We found the vant Hoff enthalpies of each mutant NKG2D-ligand interaction. We correlated observed binding energies with those predicted by a fixed-backbone RosettaDesign mutation simulation. Even simple fixed-backbone RosettaDesign provided qualitative agreement with results for 14 of 15 mutant NKG2D-ligand interactions, and quantitative agreement for 11 of those. To complete this study we plan to investigate the hydrophobic effect in the center of the site by making an additional 3 mutations (I,L, and V) at each residue.

Journal of Immunology, Apr 1, 2009

Biochemistry, Nov 24, 1999

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are ... more Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

ABSTRACT.-From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities e... more ABSTRACT.-From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities experienced unprecedented synergy. Scientific societies founded in the late eighteenth and early nineteenth century, such as the American Philosophical Society, the Academy of Natural Sciences of ...

Perspectives on Science and Christian Faith

Journal of Immunology, 2009

From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities experienced ... more From 1830 to 1850, Philadelphia's scientific, artistic, and literary communities experienced unprecedented synergy. Scientific societies founded in the late eighteenth and early nineteenth century, such as the American Philosophical Society, the Academy of Natural Sciences of Philadelphia, and the Franklin Institute, reached a stage of broad expansion. Medical science and education ballooned, and magazines, newspapers, and publishers were proliferating. The circle of the educated elite in Philadelphia was large enough to exhibit wide professional diversity, yet small enough to allow frequent associations among its members, whether scientists, artists, or both. We propose that a daguerreotype that has appeared since 1937 in several articles, often without attribution, was taken by Paul Beck Goddard in the winter of 1842-43 in the Academy of Natural Science of Philadelphia's recently constructed building at the corner of Broad and Sansom Streets. Goddard had announced as early...

ACS Symposium Series

One educational problem that technology can address is how to facilitate authentic research exper... more One educational problem that technology can address is how to facilitate authentic research experiences in the classroom for large numbers of students. The collaborative online platform GENI (Guiding Education through Novel Investigation, found at geni-science.org) provides an application that transfers protocols among institutions and into undergraduate teaching laboratories, then collects the data from students for analysis and publication. I have used this tool for several years to conduct bioinformatics research with three or four 16-student lab sections in Biochemistry I and to prepare recombinant proteins with two or three 16-student lab sections in Biochemistry II. These projects also feed into a Survey of Physical Chemistry course. Here I present technical details of how research projects have transitioned into the teaching lab. Others in the GENI consortium have accomplished similar projects in molecular biology and genetics contexts. Education researchers on our team are developing and applying assessments to shape our use of GENI as a tool. Altogether, these show that online lab protocols and data collection can be an effective way to teach students through the creative and exploratory process of asking and answering research questions.

ACS Symposium Series

In the laboratory for Survey of Physical Chemistry, students proceeded through a five-week projec... more In the laboratory for Survey of Physical Chemistry, students proceeded through a five-week project in which they measured protein-protein binding. This project engaged the students in learning physical chemistry and laboratory teachniques as they took ownership of a particular, novel protein-protein interaction. First students purified new proteins by size-exclusion chromatography and learned about separation and diffusion. Then students measured the binding strength of new protein-protein combinations using surface plasmon resonance (SPR) as they learned about SPR physics, experimental design, equilibrium binding, and data fitting using integrated rate laws. The web-based platform GENI provided protocols to the students and collected data, organizing projects spanning multiple classes. In the space of an academic year, students asked a question, then found the answer in the lab. Together, by expressing new proteins and measuring binding thermodynamics and kinetics, we found that the NKG2D immunoreceptor and its MIC ligand proteins show remarkable cross-reactivity among human, rabbit, and gorilla orthologs.