Long HCDR3s preferentially use reading frames (RF) that result in reduced hydrophobicity.

<p>(A) Peripheral blood antibody sequences from Group 1 and Group 2 healthy donors (left panel) or HIV-infected donors (right panel) were assembled into three HCDR3 length groups: (1) all HCDR3s; (2) HCDR3s of at least 24 amino acids; and (3) HCDR3s of at least 28 amino acids. (B) The percentage of sequences within each HCDR3 group using reading frame 2 of the diversity gene (RF2) was calculated for each HCDR3 length group. Non-linear regression analysis produced a sigmoidal curve of best fit (r² = 0.84). (C) Sequences that do not encode the joining gene J_H6 (top panel) or do encode J_H6 (bottom panel) were grouped by HCDR3 length and RF2 use within each HCDR3 length group was determined. The mean frequency ± SEM is shown. Non-linear regression analysis produced a sigmoidal curve of best fit. (D) The percentage of hydrophobic residues was calculated for each reading frame of every functional (lacking stop codons) diversity gene in the D2 and D3 germline gene families. The mean percentage ± SEM is shown for each reading frame. The RF2 hydrophobicity of the diversity genes which were shown to be increased in long HCDR3s are indicated by filled circles. The p values were determined using a Student’s two-tailed t-test. (E) The grand average of hydropathicity (GRAVY) was calculated for each functional reading frame of each D2 and D3 gene. A positive GRAVY score indicates hydrophobicity, and a negative GRAVY score indicates hydrophilicity. The mean GRAVY score ± SEM is shown for each reading frame. The RF2 GRAVY scores of the diversity genes that were shown to be increased in long HCDR3s are indicated by filled circles. The p values were determined using Student’s two-tailed t-test. All statistically significant differences are indicated. * = p<0.05, ** = p<0.01, *** = p<0.001.