What is the biological relevance of the specific bond properties revealed by single-molecule studies ? Philippe Robert, Anne-Marie Benoliel, Anne Pierres, Pierre Bongrand To cite this version: Philippe Robert, Anne-Marie Benoliel, Anne Pierres, Pierre Bongrand. What is the biological rel- evance of the specific bond properties revealed by single-molecule studies ?. Journal of Molecular Recognition, Wiley, 2007, 20, pp.432-447. hal-00321008 HAL Id: hal-00321008 https://hal.archives-ouvertes.fr/hal-00321008 Submitted on 12 Sep 2008 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 This un-edited manuscript was accepted and iii) is it possible to relate this for publication by the Journal of information to molecular structure ? Molecular Recognition and published in volume 20, pp 432-447. It was published Keywords : laminar flow chamber, atomic on line on august 28, 2007 by Wiley force microscopy, biomembrane force probe, optical traps, surface-attached InterScience ( www.interscience.wiley.com molecules, binding strength, unbinding - DOI 10.1002/jmr.827) force, off-rate, Bell’s law 1 - INTRODUCTION What is the biological relevance of During the last decade, simultaneous the specific bond properties development of several methods allowing to revealed by single-molecule monitor bond formation and dissociation at the studies ? single molecule level yielded a new kind of information on molecular behaviour. The aim Philippe Robert 1,2,3 , Anne-Marie of this review is to discuss the input of this Benoliel 1,2,3 , Anne Pierres 1,2,3 and Pierre advance to current biological wisdom. For the Bongrand 1,2,34 sake of clarity and homogeneity, we shall focus on proteins. 1 INSERM UMR600, 2 CNRS UMR 6212, 3 Université de la Mediterranée Importance of molecular interactions. Life is based on molecular interactions. Indeed, considering proteins, enzymes need to bind specifically to substrates they will During the last decade, many authors took maintain in close proximity with adequate advantage of new methodologies based on reagents. Scaffolding proteins bind to each atomic force microscopy, biomembrane other in order to maintain the cell architecture. force probes, laminar flow chambers or Signalling is based on the formation of optical traps to study at the single molecule multimolecular complexes that will be level the formation and dissociation of generated through dedicated binding sites bonds between receptors and ligands frequently borne by widely spread structures attached to surfaces. Experiments provided such as SH2, SH3 or plecskstrin homology a wealth of data revealing the complexity of domains. Membrane receptors bind to specific bond response to mechanical forces and the ligands. Membrane adhesion receptors are a dependence of bond rupture on bond prominent example: nearly all steps of cell life history. These results supported the including survival, proliferation, existence of multiple binding states and/or differentiation, migration or activation are reaction pathways. Also, single bond studies heavily dependent on adhesive interactions. It allowed us to monitor attachments mediated is therefore not surprising that between 25 % by a few bonds. The aim of this review is to and 50 % of cell membrane molecules may discuss the impact of this new information well be adhesion receptors (Barclay, 1998). on our understanding of biological Also, it was stated in standard treatises that molecules and phenomena. The following “the biological functions of proteins almost points are discussed: i) which parameters do invariably depend on their direct physical we need to know in order to predict the interaction with other proteins” (Creighton, behaviour of an encounter between 1993). receptors and ligands, ii) which information Thus, it is not surprising that most is actually yielded by single-molecule studies biologists are deeply interested in molecular recognition and biomolecule interaction. However, while these phenomena are taught in 4 Corresponding author: Pr. Pierre Bongrand, every textbook and may be felt as well Laboratoire “Adhésion et Inflammation”, INSERM understood, several lines of research that UMR600, Parc de Luminy, Case 937, 13288 steadily increased during the last decade Marseille Cedex 09, France – Email : [email protected] 2 brought dramatic changes to our understanding Kramers (1940) to account for the rate of of molecular interactions. molecular reactions and basic analysis of the effect of force and attachment to surfaces Recent progress in studying molecular (Bell, 1978) was revived (Evans and Ritchie, interactions . 1997). Older reports formed the basis of a At least five major advances led to dramatic recent surge of theoretical analyses that are conceptual advances. intended to process and interpret currently i) extensive use of structural studies available experimental data. based on crystallography or nuclear magnetic v) Simultaneously, computer resonance yielded an increasing number of simulation evolved as a new approach for available molecular complexes known with understanding as well as predicting the angström resolution. Thus, we probably know behaviour of biomolecules. While thirty years fairly well what protein-protein binding ago, it appeared as a remarkable feat to interfaces look like (Lo Conte et al., 1999). It simulate the behaviour of a few tens of water seems a reasonable order of magnitude to molecules in a box (Dashevsky and Sarkisov, assume that the association of two protein 1974), it becomes feasible to simulate molecules will make several hundreds of interactions between proteins comprising squared angströms inaccessible to water, with thousands of atoms (Schueler-Furman et al., several tens of close interatomic contacts. 2005 ; Gray, 2006 ; Rueda et al., 2007) and ii) In addition to descriptive mimic force-induced bond rupture as studied information, systematic use of site-directed experimentally on models such as streptavidin- mutagenesis following the pioneering study of biotin interaction (Zhou et al., 2006). Cunningham et al. (1989) opened the Thus, it seems reasonable to examine possibility to assess the quantitative the impact of aformentioned lines of research importance of the interactions that were "seen". on the conceptual framework we should use to The basic principle consisted of systematically consider biomolecule interactions. replacing individual aminoacids constituting the binding surface with alanine and measuring An agenda for studying molecular the influence of this change on affinity. This interactions . approach yielded information on the influence A biologist willing to explore and understand on affinity of individual hydrogen bonds or the molecular interactions underlying cell ionic interactions. function will need the following three kinds of iii) Following the pioneering work of information. Tha et al. (1986) and Evans et al. (1989), several innovative methods were i) A first requirement consists of defining a set systematically used to monitor individual bond of quantitative parameters that are both formation and dissociation between surface- necessary and sufficient to account for attached biomolecules subjected to controlled molecular behaviour when they have been force in the piconewton range. These include measured. As an example, it has long been laminar flow chambers (Kaplanski et al., 1993 considered that the affinity constant might ; Alon et al., 1995 ; Pierres et al., 1996), account for most aspects of biomolecule atomic force microscopy (Florin et al., 1994 association. ;Lee et al., 1994 ; Hinterdorfer et al., 1996), optical tweezers (Miyata et al., 1996 ; Rinko et ii) Once useful parameters have been defined, al., 2004 ; Litvinov et al., 2005) biomembrane it is necessary to elaborate efficient methods force probes (Evans et al., 1994 ; Merkel et al., for measuring them. In fact, the development 1999). In addition to unprecedented accuracy, of powerful ways of studying individual bonds these methods yielded qualitatively new was certainly an incentive to look for more information such as bond response to accurate ways of describing and understanding mechanical forces. these bonds. iv) The need to interpret the new kind of information yielded by aforementioned iii) Finally, it is certainly useful to obtain methods was an incentive to reexamine the quantitative links between molecular structure physical basis of molecular interaction. Thus, and interaction properties. First, this would pioneering work initiated by Eyring (1935) and allow us to predict interaction parameters 3 between known molecules without a need to be considered. Here are two important perform lengthy and delicate experiments. examples. Second, this would markedly improve our A prominent step in the development understanding of biological phenomena. Thus, of immune responses is the interaction of T an important effort was done in building so- lymphocytes and antigen presenting cells called docking algorithms intended to predict exposing on their membranes cognate the interaction between