Dynamics of Biomedical Molecules in Vision, Allostery, and Folding David Kliger, Chemistry & Biochemistry The Kliger research group uses time-resolved spectroscopy to study protein reactions.  Proteins are central to health, making study of their structure and function critical.  While traditional biochemical methods provide insight about the later steps in function, modern optical methods (particularly pulsed lasers and polarized light) reveal previously unknown steps occurring on a short time scale important in biomedical processes providing information that can lead to the development of new or more effective drug therapies. (Keywords: physical chemistry, biophysics, time-resolved spectroscopy)

David Kliger and his colleagues initiate reactions with nanosecond laser pulses and follow the course of reaction on subsequent time scales using UV/visible absorbance measurements.  In addition, state of the art polarization techniques provide unique structural insight about transient species.  Among the reactions that interest Kliger's research group are those involved in the activation of the visual pigment rhodopsin, those of various heme proteins, as well as those that enable proteins to fold into their native structures.

In addition to its important role in the process by which we see the dimmest light, rhodopsin is a model system for understanding G protein-coupled receptors (GPCRs). GPCRs represent one of the largest families in the human genome, constituting approximately 5% of our genes.  GPCRs bind to and transduce signals for a large variety of ligands, including neurotransmitters, odorants, and hormones. Given their wide prevalence it is not surprising that they mediate the actions of over 50% of the medicines used to treat disorders as diverse as cardiovascular disease, drug dependency, and mental illness.  Since most of these medicines were developed serendipitously before it was even understood what GPCRs were, it can conservatively be predicted that GPCR understanding will revolutionize medical treatment.

Mechanisms of most GPCRs are difficult to study because concentrations of many GPCRs occuring in our bodies are low and because the vast majority of GPCR's activation kinetics are controlled by ligands which diffuse to them.  Rhodopsin, however, has an endogenous ligand that is triggered by light absorption. This makes it possible to study the rhodopsin activation mechanism step by step, from its initial triggering through to final activation.

Hemoglobin, Ligands and Allosteric Mechanisms

The Kliger laboratory has used time-resolved circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy to illuminate the multi-step nature of allostery in human hemoglobin. Allostery down regulates the ligand-binding strength of the R quaternary state in the T state, enabling efficient off-loading of oxygen to the tissues. Time-resolved CD and MCD of the tryptophan amino acid residues showed that photolysis of ligands from the R state first triggers formation of the Trp b37-Asp a94 hydrogen bond, pictured here in the "hinge" region of the interface that connects the two dimers of the hemoglobin tetramer. This step, in turn, triggers a larger structural change several microseconds later, completing conversion of the R structure to T. This kinetic result helps to explain why Trp b37, which is highly conserved in the sequences of vertebrate hemoglobins, plays such a key role in cooperativity and allostery.

Understanding Principles of Protein Folding

The processes by which proteins fold to their native structures are of fundamental interest to biophysicists. This problem has taken on even more significance in recent years. First, a number of diseases have been identified with misfolded proteins. Second, the human genome project has opened doors to the identification of many new proteins important to our physiology. As their sequences are found, accurate prediction of corresponding protein structures will be a major advantage for biomedical investigators as they attempt to determine the function of these proteins and design therapeutic remedies for related pathological conditions. In order to do this, however, scientists will first need to understand the fundamental mechanisms by which proteins fold.

Using time-resolved laser spectroscopy, the Kliger group has triggered protein folding with techniques such as fast ligand photodissociation, fast electron transfer, and fast laser temperature jumps. Combined with a variety of spectroscopic probes, they have gained new insights into the early events in protein folding which drive these macromolecules to take on their native structures.

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