SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

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The IgG CH2 domain continues to hold promise for the development of new therapeutic entities because of its bifunctional role as a biomarker and effector protein. The need for further understanding of molecular stability and aggregation in therapeutic proteins has led to the development of a breakthrough quantum cascade laser microscope to allow for real-time comparability assessment of an array of related proteins in solution upon thermal perturbation. Our objective was to perform a comprehensive developability assessment of three similar monoclonal antibody (mAb) fragments: CH2, CH2s, and m01s. The CH2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. CH2s has a 7-residue deletion at the N-terminus and a 16-residue C-terminal extension containing a histidine tag. The m01s construct is identical to CH2s, except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 °C for all three proteins in an array. Co-distribution and two-dimensional infrared correlation spectroscopies yielded the mechanism of aggregation and stability for these three proteins. The level of detail is unprecedented, identifying the regions within CH2 and CH2s that are prone to self-association and establishing the differences in stability. Furthermore, CH2 helical segments, β-sheets, β-turns, and random coil regions were less stable than in CH2s and m01s because of the presence of the N-terminal 310-helix and β-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.

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Functional protein-protein interactions are essential for many physiological processes. Some of these functional interactions have been hypothesized to play a role in substrate channeling, cofactor or coenzyme transfer, and compartmentation in glycolysis as a result of transient or dynamic interactions between glycolytic enzymes. Herein, Brownian dynamics (BD) elucidates the interactions between the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH); the transfer of the cofactor nicotinamide adenine dinucleotide (NAD) between LDH and GAPDH. BD tests the hypotheses of whether the interaction between GAPDH and LDH produces a functional complex that can efficiently and reversibly transfer the cofactor NAD(H) between both enzymes. Preliminary results suggest favorable enzyme-enzyme complexes between GAPDH and LDH involving four different binding modes. These complexes are mainly stabilized by positively charged lysine residues and negatively charged glutamates and aspartates from both GAPDH and LDH. The efficiency of transfer determined as the relative number of BD trajectories that reached any active site of LDH or GAPDH, show higher transfer efficiencies (about an order of magnitude) when the cofactor NAD is transferred from a GAPDH active site to an LDH active site as compared to transfer efficiencies of NAD from solution to each enzyme of the complex. The average transfer time of NAD from solution to the free enzymes is 500 ns as compared to 57-200 ns when NAD is transferred between active sites of a GAPDH/LDH complex. Similarly, the frequency distribution profiles of transfer times suggest a preference for channeling NAD between GAPDH and LDH as compared to diffusing from solution. Channeling transfer is more efficient that solution transfer, due to active site proximity, favorable electrostatics and complex geometry