several groups have used surface-enhanced Raman spectroscopy (SERS) and surface-enhanced resonance Raman spectroscopy (SERRS) to characterize biological molecules. Koglin and Séquaris conducted in-depth investigations of nucleic acids and DNA with silver colloids ref 1 and references therein.Several amino acids have been also studied by using silver colloids to obtain SERS.2"6 The aromatic amino acids, tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), and histidine (His), exhibited intense spectra2"4 but displayed great variations in relative peak intensities and frequencies from study to study. Methods

Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric protein...

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.

Two naturally occurring mutants of human lecithincholesterol acyltransferase (LCAT), T123I and N228K, were expressed in COS-1 and Chinese hamster ovary cells, overproduced, and purified to homogeneity in order to study the structural and functional defects that lead to the LCAT deficiency phenotypes of these mutations. The mutants were expressed and secreted by transfected cells normally and had molecular weights and levels of glycosylation similar to wild type LCAT. The purified proteins (>98% purity) had almost indistinguishable structures and stabilities as determined by CD and fluorescence spectroscopy. Enzymatic activities and kinetic analysis of the pure enzyme forms showed that wild type LCAT and both mutants were reactive with the water-soluble substrate, p-nitrophenyl butyrate, indicating the presence of an intact core active site and catalytic triad. Both the T123I and N228K mutants had markedly depressed reactivity with reconstituted HDL (rHDL), but T123I retained activity with low density lipoprotein. To determine whether defective binding to rHDL was responsible for the low activity of both mutants with rHDL, the equilibrium binding constants were measured directly with isothermal titration calorimetry and surface plasmon resonance (SPR) methods. The results indicated that the affinities of the mutants for rHDL were only about 2-fold lower than the affinity of wild type LCAT (K d ‫؍‬ 2.3 ؋ 10 ؊7 M). Together, the activity and equilibrium binding results suggest that the T123I mutant is defective in activation by apolipoprotein A-I, and the N228K mutant has impaired binding of lipid substrate to the active site. In addition, the kinetic binding rate constants determined by the SPR method indicate that normal LCAT dissociates from rHDL, on average, after one catalytic cycle.

Total internal reflection fluorescence was used to analyze rabbit immunoglobulin G (IgG) nonspecifically adsorbed on thin nylon f i i . Fluorescence energy transfer measurements were recorded for two comparative systems: (1) adsorbed fluorescein isothiocyanate (FITC) tagged IgG and tetramethylrhodamine isothiocyanate (TRITC) tagged nylon; (2) FITC labeled IgG and TRITC labeled anti-rabbit IgG (anti-IgG). These experiments were used to examine the nylon/IgG and IgG/solution interfaces, respectively, as a function of the surrounding buffer pH and washing conditions. Results were correlated with IgG mass loading measured by the bicinchoninic acid (BCA) method and anti-IgG binding activity assessed using TRITC-tagged anti-IgG. When washed with pH 6.9 phosphate buffer, the amount of adsorbed IgG increased with the concentration of IgG used in the adsorption step. Fluorescence data were consistent with an IgG bilayer model, where the upper layer did not interact with the TRITC labeled nylon surface but bound to and exchanged with solution borne anti-IgG. After washing with carbonate buffer at pH 8.7, only a tightly adsorbed, unexchangeable layer of IgG remained on the nylon surface, with mass corresponding to less than a monolayer. In a pH 8.7 buffer, no energy transfer took place between adsorbed FITC labeled IgG and the TRITC tagged anti-IgG although anti-IgG did adhere to the nylon, suggesting coadsorption of anti-IgG rather than immunochemical binding. When the buffer was replaced by a pH 6.9 phosphate buffer, energy transfer was observed, indicating that binding had occurred.

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