Stopped flow system for FTIR difference spectroscopy of biological macromolecules

Masuch, Ralf; Moss, David A.

doi:10.1007/978-94-011-4479-7_309

Cited by 5 publications

(3 citation statements)

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“…(1) New methods to perturb proteins or to initiate protein reactions will expand the number of systems that can be investigated with reaction-induced IR difference spectroscopy and will also pave the way for biotechnology applications. Of particular interest here are a number of mixing devices that have recently been developed (White et al 1995 ;Fahmy, 1998Fahmy, , 2001Masuch & Moss, 1999 ;Hinsmann et al 2001 ;Kauffmann et al 2001) and which aim at making IR spectroscopy as universally applicable as UV/visible spectroscopy. (2) New applications of IR spectroscopy and IR imaging in biomedicine (reviewed by Jackson & Mantsch, 1996 ;Jackson et al 1997 ;Naumann, 2001).…”

Section: Discussionmentioning

confidence: 99%

What vibrations tell about proteins

Barth

Zscherp

2002

Quart. Rev. Biophys.

1,857

123

2,185

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This review deals with current concepts of vibrational spectroscopy for the investigation of protein structure and function. While the focus is on infrared (IR) spectroscopy, some of the general aspects also apply to Raman spectroscopy. Special emphasis is on the amide I vibration of the polypeptide backbone that is used for secondary-structure analysis. Theoretical as well as experimental aspects are covered including transition dipole coupling. Further topics are discussed, namely the absorption of amino-acid side-chains, 1H/2H exchange to study the conformational flexibility and reaction-induced difference spectroscopy for the investigation of reaction mechanisms with a focus on interpretation tools.

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Section: Discussionmentioning

confidence: 99%

What vibrations tell about proteins

Barth

Zscherp

2002

Quart. Rev. Biophys.

1,857

123

2,185

View full text Add to dashboard Cite

show abstract

“…This is because it is difficult to make a concentrated and thus highly viscous protein solution flow rapidly through the thin slit between the windows of an infrared cuvette. Nevertheless, successful developments of a stopped-flow apparatus for infrared spectroscopy have been reported ( , ). As the components have to withstand high pressure and since small dead volumes are desired, HPLC technology was used for the interconnections between the conventional pneumatic flow-inducing system and the mixing and cuvette assembly.…”

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confidence: 99%

Reaction-Induced Infrared Difference Spectroscopy for the Study of Protein Reaction Mechanisms

2001

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This paper reviews state-of-the-art reaction-induced infrared difference spectroscopy of proteins. This technique enables detailed characterization of enzyme function on the level of single bonds of proteins, cofactors, or substrates. The following methods to initiate a reaction in the infrared sample are discussed: (i) light-induced difference spectroscopy, (ii) attenuated total reflection with buffer exchange, (iii) the infrared variant of stopped and continuous flow, (iv) temperature and pressure jump, (v) photolytical release of effector substances from caged compounds, (vi) equilibrium electrochemistry, and (vii) photoreduction. Illustrating applications are given including hot topics from the fields of bioenergetics, protein folding, and molecule--protein interaction.

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“…This avoids the comparison of spectra from different samples and provides a very sensitive detection of the small infrared absorbance changes associated with protein reactions. Several methods are available to trigger the binding of molecules to proteins: rapid mixing in specially designed cuvettes [50,70], the use of photolabile molecules that release the substrate upon illumination [14], and several techniques using attenuated total reflection (ATR) [33]:…”

Section: Introductionmentioning

confidence: 99%

Two sides of the same coin: How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase

Barth

2016

BSI

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Abstract. This review summarises our infrared spectroscopy and density functional theory studies on the mutual interactions between enzymes and their substrates. We investigated phosphoenolpyruvate bound to pyruvate kinase (EC 2.7.1.40, M1 isozyme), ATP bound to the Ca 2+ -ATPase (SERCA1a), and the aspartylphosphate moiety of the Ca 2+ -ATPase phosphoenzyme E2P. Conformational changes of the enzymes and distortions of substrate structure are discussed. In all cases, the infrared absorption of the substrate in the enzyme environment could be identified by a combination of reaction-induced difference spectroscopy and isotopic labelling. The experimentally-determined vibrational frequencies were interpreted in structural terms using experimental correlations or modelling of the active site in density functional theory calculations. For none of the three systems, a weakening of the bond that is cleaved in the following enzymatic reaction could be detected in the ground state of the enzyme-substrate complex. However, for the dephosphorylation reaction of the Ca 2+ -ATPase phosphoenzyme E2P, a high energy intermediate, not detected in experiments, is the reactant state according to density functional theory calculations.

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Stopped flow system for FTIR difference spectroscopy of biological macromolecules

Cited by 5 publications

References 1 publication

What vibrations tell about proteins

What vibrations tell about proteins

Reaction-Induced Infrared Difference Spectroscopy for the Study of Protein Reaction Mechanisms

Two sides of the same coin: How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase

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