The C␣OH⅐⅐⅐O hydrogen bond has been given little attention as a determinant of transmembrane helix association. Stimulated by recent calculations suggesting that such bonds can be much stronger than has been supposed, we have analyzed 11 known membrane protein structures and found that apparent carbon ␣ hydrogen bonds cluster frequently at glycine-, serine-, and threonine-rich packing interfaces between transmembrane helices. Parallel righthanded helix-helix interactions appear to favor C␣OH⅐⅐⅐O bond formation. In particular, C␣OH⅐⅐⅐O interactions are frequent between helices having the structural motif of the glycophorin A dimer and the GxxxG pair. We suggest that C␣OH⅐⅐⅐O hydrogen bonds are important determinants of stability and, depending on packing, specificity in membrane protein folding.T he hydrogen bond is a key element in the interplay between stability and specificity in protein folding. The desolvation penalty associated with burial of polar side chains in an aqueous environment is not always fully recovered by hydrogen bond formation, so hydrogen bonds provide a small or even unfavorable net energy contribution to folding. However, the strength and directionality of hydrogen bonds make them an important factor in discriminating between correctly folded and misfolded states. Hence, polar interactions tend to contribute more to specificity than to stability in soluble proteins (1-3). Conversely, in the apolar environment of biological membranes donor and acceptor groups cannot be satisfied by the solvent, and hydrogen bonds strongly stabilize the helical conformation of membrane spanning domains (4) and can stabilize tertiary interactions as well (5-9). We are interested in the role of hydrogen bonds in the association of transmembrane helices, a stage that is pivotal in the folding of membrane proteins (4). Recently, the DeGrado group and our laboratory showed that the substitution of a single polar amino acid residue into model transmembrane helices induces homooligomerization (10 -13); the association driven by hydrogen bonding can be strong and independent of packing details. Thus, in the apolar environment, the strength of hydrogen bonds can stabilize the association of transmembrane helices, although a lack of a need for sequence specificity could create a danger of inducing promiscuous association (10, 13).Weaker hydrogen bonds, such as those between carbon and oxygen atoms (COH⅐⅐⅐O), have received little attention in the membrane protein field, and their occurrence in membrane proteins has never been surveyed. The C␣ is an activated carbon donor because it is bound to the electron-withdrawing amide NOH and CAO groups, and, in soluble proteins, hydrogen bonds between main-chain C␣OH groups and backbone or side-chain oxygen atoms are often observed (14-17). Despite its abundance, the structural contribution of the C␣OH⅐⅐⅐O hydrogen bond has been unclear and its interaction energy has been believed to be small. Recently, by using ab initio calculations, Vargas et al. (18) and Scheiner et al. (19...