References 1
Baldwin RL 2002 Making a networkofhydrophobicclusters. Science 295 487-489 Brandts JF, Hu CQ, Lin LN 1989 A simple model for proteins with interacting domains. Applications to scanning calorimetrydata. Biochemistry 28 8588-8596 Conejero-Lara F, Mateo PL, Aviles FX, Sanchez-Ruiz JM 1991 Effect of Zn2 on the thermal denaturation ofcarboxypeptidase B. Biochemistry 30 2067-2072 Cooper A 1976 Thermodynamic fluctuations in protein molecules. Proc Natl Acad Sci USA 73 2740-2741 Cooper A 2005 Heat...
Structure Orientation and Possible Mechanism of Action of Therapeutic Agents
Other applications of IRRAS follow from the expectation that understanding the molecular mechanism of surfactant function will improve rational design of therapeutic agents for respiratory distress syndrome. Johansson et al. 2001 recently reviewed the use of artificial surfactants based on analogues of SP-B and SP-C. For example, peptides with at least 17 amino acids corresponding to the C-ter-minal part of SP-B accelerate surfactant spreading and improve lung compliance in fetal rabbits. Also...
D Maps of Homopolypeptides
Proteins are very complex entities that can undergo complex changes after being exposed to an external perturbation. Homopolypeptides have been used extensively to assign IR bands to secondary structures Susi 1969 . ttey are used because of their ability to adopt regular-canonical conformations without containing tertiary structure. Polyproline is a particular example since its inability to form hydrogen bonds with other peptide bonds produces the so-called polyproline I and polyproline II PPII...
TROSY for Studies of Intermolecular Interactions and Drug Design
Study of intermolecular interactions, including protein-protein, protein-nucleic acid, and protein-ligand interactions, can provide valuable information related to the physiological roles of a newly discovered protein. Moreover, these studies are of primary interest for drug discovery aiming at the design of high-affinity ligands for relevant biological molecules Hajduk et al. 1999 Pellecchia et al. 2002 Meyer and Peters 2003 . Several standard NMR experiments used to study intermolecular...
Liquid Mixing in Small Volumes and Microfluidics
Two consecutive processes dominate the mixing of liquids fusion of the liquid boundaries and diffusion of the components. Although both processes are similar in bulk fluid and microfluidics, their outcome is significantly different unexpected mixing results in microfluidics were reported. Two liquid streams can flow alongside in a tube a few micrometers wide over a period of time without mixing, almost as if they were separated by glass Knight 2002 . tte fusion step is not only the first step,...
TROSY for Observation of Scalar Couplings Across Hydrogen Bonds
Hydrogen bonds play a key role in the structure and function of biological mac-romolecules. tteir presence is usually only indirectly inferred from experimental data, e.g., from NOEs and proton exchange rates, or from analysis of refined 3D structures based on geometrical considerations. Direct detection of hydrogen bonds in proteins and oligonucleotides in NMR spectra was enabled by the recent observation of scalar spin-spin couplings across hydrogen bonds Dingley and Grzesiek 1998 Cordier and...
What Is DSC
DSC is a powerful technique to characterize temperature-induced conformational changes in proteins and other biological macromolecules. A differential scanning calorimeter basically consists of two cells the reference cell and the sample cell for a detailed technical description of this type of calorimeter, see Privalov 1980 Plotnikov et al. 1997 . ttis section is concerned with the case in which the sample is a diluted protein solution and the reference is the pure solvent buffer . Both cells...
LAURDAN in Cell Membranes and Tissues
From the pioneering work of Yu et al. 1996 and Parasassi et al. 1997 LAURDAN was proposed to be very promising in exploring cell membranes. In these studies domains of sizes below, in the same range as and above the microscope resolution limit 0.3 m were observed in OK cells, red blood cells and brush-border native membranes, respectively Parasassi et al. 1997 . tte LAURDAN GP differences observed in compositionally complex mixtures and artificial lipid ternary mixtures containing...
What Is Heat Capacity
A DSC calorimeter provides a profile of heat capacity versus temperature for a protein solution. In any textbook of thermodynamics or physical chemistry the reader will find a definition of heat capacity as the temperature-derivative of the enthalpy or energy function, ttis definition admits a simple and intuitive interpretation heat capacity is a measure of the capacity of the system to store energy, ttat is, whenever there is in the system an efficient energy storage mechanism, the energy of...
Selective IsotopeLabeling Strategies
Selective or specific labeling techniques are outstandingly useful tools for NMR studies of large proteins Gardner and Kay 1998 Goto and Kay 2000 . One important application is the reduction of the number of resonances in overcrowded NMR spectra, where only signals from an individual amino acid or from a group of amino acids in the sequence can then be observed. Several protocols have been developed for residue-specific labeling of proteins in a protonated background based on E. coli expression...
Cryoelectron Tomography
So far, for obtaining a 3D reconstruction of a particular biological complex, we have isolated it from the rest of the cell components and have averaged images of thousands of copies. In cryo-EM the final 3D maps reflect the near-native state of the molecules in vitro. Obviously, the real native conditions should include the biological context and the uniqueness of each object. Up to now, this task has seemed unreachable. But a new exciting era is opening, in which several groups are already...
The Effect ofa Mechanical Force on theThermodynamics and Kinetics ofthe Protein
Considerations from ermodynamics and Statistical Mechanics Equilibrium vs. Nonequilibrium Unfolding Classical thermodynamics equilibrium thermodynamics or thermodynamics for short deals with quasistatic processes, which are idealized, infinitely slow processes that can be approximated in practice by performing them very slowly. ttus, thermodynamics is not concerned with the rate at which a process takes place i.e., kinetics . Time-dependent thermodynamic processes are studied by nonequilibrium...
Implementation ofTROSY 2D [15N1HTROSY
For the implementation of TROSY into experimental schemes it is essential that the scalar couplings between participating nuclei are not decoupled, and that only the most slowly relaxing multiplet component is selected for detection, while all other components are suppressed Fig. 5.2 . Here, we discuss the simplest scheme for the application of the TROSY principle the 2D heteronuclear single-quantum correlation HSQC experiment 15N,1H -TROSY Fig. 5.3a which is based on the Fig. 5.3. Experimental...
ForceClamp SMFS
As we have seen in the previous section, mechanical unfolding experiments are typically performed at a constant length not at a constant force , where it is not possible to directly calculate the unfolding rate constants as a function of the force. However, it is now possible to stretch the molecule at a constant force and directly obtain the kinetic parameters of the process in a model-independent manner Oberhauser et al. 2001 . ttese force-clamp experiments have so far been performed on a few...
SingleMolecule Mechanics vs Bulk Biochemical Assays
Until recently, the only way to measure the stability of a protein was to change its physical temperature or pressure or chemical environment using concentrated solutions of guanidinium chloride or urea, acid or alkaline pH and monitor the loss of protein conformation by spectroscopic techniques in order to determine the change in the Gibbs free energy. Most of these folding studies have been performed using chemical d naturants acting on untethered proteins i.e., in solution . However, a...
LAURDAN Probe the Tips
As is the case with the vast majority of UV-excited fluorescent probes, confocal or epifluorescence microscopy experiments are difficult to perform with LAURDAN. As already mentioned, the extent of LAURDAN photobleaching under epifluorescence and confocal microscopy is severe, making it almost impossible to collect images on LAURDAN-labeled specimens for more than a few seconds Bagatolli and Gratton 2001 . A way to circumvent this problem is by using two-photon-excitation fluorescence...
TROSY CRIPT and CRINEPT for Studies of Very Large Structures
With increasing molecular mass, most signal is lost during polarization transfer periods in heteronuclear NMR experiments where TROSY is not active. If the conventional polarization transfer element INEPT Morris and Freeman 1979 Burum and Ernst 1980 is replaced by CRINEPT Riek et al. 1999, 2002 and CRIPT Dalvit 1992 , the signal loss can be reduced. Using NMR experiments based on TROSY and CRINEPT or CRIPT, we can now investigate macromolecular systems with molecular masses up to 900 kDa by...
References Ula
Ainavarapu SRK, L L, Badilla CL, FernandezJM 2005 Ligand binding modulatesthe mechanical stability ofdihydrofolate reductase. Biophys J 89 3337-3344 Alberts B 1998 The cell as a collection of protein machines preparing the next generation of molecular biologists. Cell 92 291-294 Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P 2002 The molecular biology of the cell, 4th edn. Garland Science, NewYork, p 1103 Albrecht C, Blank K, Lalic-Multhaler M, Hirler S, Mai T, Gilbert I, Schiffmann...
The Calorimetric Criterion for TwoState Behaviour
tte two-state equilibrium model assumes that only two states of the protein are significantly populated at equilibrium the native and the unfolded reversibly denatured states, tte unfolded state and, to some extent, the native state as well is to be considered as an ensemble of conformations in equilibrium. For this reason, these states are often referred to as macrostates. tte two-state equilibrium model has been extensively used in studies on protein folding and stability. In fact, it has...
Polyprotein Strategy Protein Repeats as SingleMolecule Fingerprints Linkers and
As we have seen Sect. 8.3.2 , proteins tend to denature to some extent when adsorbed onto a surface, and for this reason long modular proteins are very good systems for SMFS studies. Such proteins are made up of several modules and, thus, two of these can readily be partially or totally sacrificed for the required attachments of the molecule to the cantilever tip and to the substrate. Furthermore, the proximal region to the substrate of a force-extension curve is often the main source of...
Creating and Dispensing Small Liquid Volumes
tte controlled dispensing of very small liquid volumes was first demonstrated by Elmqvist in the context of printing in the Siemens-Elema Minograf recording mechanism US patent 2,566,443, issued September 1951 . Important factors in the dispensing of small liquids volumes are - Dynamic range of the dispensed volume - Dispensing frequency determines throughput - Ease of operation and maintenance - General compatibility of surfaces and liquids compatible with labile compounds tte preferred size...