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How membrane proteins distribute and behave on the surface of cells depends on the molecules' chemical potential. However, measuring this potential, and how it varies with protein-to-protein distance, has been challenging. Here, we present a method we call hydrodynamic trapping that can achieve this. Our method uses the focused liquid flow from a micropipette to locally accumulate molecules protruding above a lipid membrane. The chemical potential, as well as information about the dimensions of the studied molecule, are obtained by relating the degree of accumulation to the strength of the trap. We have used this method to study four representative proteins, with different height-to-width ratios and molecular properties; from globular streptavidin, to the rod-like immune cell proteins CD2, CD4 and CD45. The data we obtain illustrates how protein shape, glycosylation and flexibility influence the behaviour of membrane proteins, as well as underlining the general applicability of the method.

Release of halide ions is an essential step of the catalytic cycle of haloalkane dehalogenases. Here we describe experimentally and computationally the process of release of a halide anion from the buried active site of the haloalkane dehalogenase LinB. Using stopped-flow fluorescence analysis and umbrella sampling free energy calculations, we show that the anion binding is ion-specific and follows the ordering I(-) > Br(-) > Cl(-). We also address the issue of the protonation state of the catalytic His272 residue and its effect on the process of halide release. While deprotonation of His272 increases binding of anions in the access tunnel, we show that the anionic ordering does not change with the switch of the protonation state. We also demonstrate that a sodium cation could relatively easily enter the active site, provided the His272 residue is singly protonated, and replace thus the missing proton. In contrast, Na(+) is strongly repelled from the active site containing the doubly protonated His272 residue. Our study contributes toward understanding of the reaction mechanism of haloalkane dehalogenase enzyme family. Determination of the protonation state of the catalytic histidine throughout the catalytic cycle remains a challenge for future studies.